Selenium atom on phosphate enhances specificity and sensitivity of DNA polymerization and detection

2021 ◽  
Author(s):  
Guang-Cheng Luo ◽  
Jun Zhang ◽  
Mei Yang ◽  
Hongfei He ◽  
Zhen Huang
Keyword(s):  
Nucleic Acid ◽  
High Specificity ◽  
Nucleic Acid Detection ◽  
Selenium Atom ◽  
Dna Polymerization ◽  
Specificity And Sensitivity ◽  
Advanced Applications

DNA polymerization is of high specificity in vivo. However, its specificity is much lower in vitro, which limits the advanced applications of DNA polymerization in ultrasensitive nucleic acid detection. Herein...

The Toxicity Testing of Cyanobacterial Toxins In Vivo and In Vitro by Mouse Bioassay: A Review

2021 ◽  
Vol 21 ◽  
Author(s):  
Hamed Ahari ◽  
Bahareh ‎ Nowruzi ◽  
Amir Ali Anvar ◽  
Samaneh Jafari Porzani
Keyword(s):  
Toxicity Testing ◽  
High Specificity ◽  
Immunological Methods ◽  
Water Bloom ◽  
Mouse Bioassay ◽  
Cell Extracts ◽  
Specificity And Sensitivity ◽  
Main Technique

: Different biological methods based on bioactivity are available to detect cyanotoxins, including neurotoxicity, immunological interactions, hepatotoxicity, cytotoxicity, and enzymatic activity. The mouse bioassay is the first test employed in laboratory cultures, cell extracts, and water bloom materials to detect toxins. It is also used as a traditional method to estimate the LD50. Concerning the ease of access and low cost, it is the most common method for this purpose. In this method, a sample is injected intraperitoneally into adult mice, and accordingly, they are assayed and monitored for about 24 hours for toxic symptoms. The toxin can be detected using this method from minutes to a few hours; its type, e.g., hepatotoxin, neurotoxin, etc., can also be determined. However, this method is nonspecific, fails to detect low amounts, and cannot distinguish between homologues. Although the mouse bioassay is gradually replaced with new chemical and immunological methods, it is still the main technique to detect the bioactivity and efficacy of cyanotoxins using LD50 determined based on the survival time of animals exposed to the toxin. In addition, some countries oppose animal use in toxicity studies. However, high cost, ethical considerations, low-sensitivity, non-specificity, and prolonged processes persuade researchers to employ chemical and functional analysis techniques. The qualitative and quantitative analyses, as well as high specificity and sensitivity, are among the advantages of cytotoxicity tests to investigate cyanotoxins. The present study aimed at reviewing the results obtained from in-vitro and in-vivo investigations of the mouse bioassay to detect cyanotoxins, including microcystins, cylindrospermopsin, saxitoxins, etc.

Reflections of Fibrin Formation by In Vitro Testing

1979 ◽  
Author(s):  
G. Wilner
Keyword(s):  
Gel Permeation Chromatography ◽  
High Specificity ◽  
Structural Data ◽  
Specificity And Sensitivity ◽  
Immunological Tests ◽  
Great Specificity ◽  
Technical Simplicity ◽  
Derivatives Of

Measurement of enzymatically-modified fibrinogen, fibrin, or related proteolysie products represent a means whereby the action of several enzymes can be quantiated both in vitro and in vivo. Advances in physicochemical techniques and in immunology over the past decade permit translation of recently acquired primary structural data on fibrinogen and fibrin into sensitive and specific assays for detecting derivatives of these proteins in the circulation. Physicochemical techniques that have been used by various investigators include paracoagulation tests, affinity chromatography, gel permeation chromatography, and electrophoresis. The limitations in specificity and sensitivity of these techniques in detecting circulating fibrin(ogen) derivatives is offset by the technical simplicity of most of these procedures, and perhaps these techniques in combination may ultimately yield useful clinical tests. Immunological tests can, in theory, offer vastly improved specificity and sensitivity as compared with the above mentioned procedures. However, preparation of immune reagents of high specificity is both tedious and difficult, usually requiring multiple adsorbtion steps to harvest the desired antibody population. Our recent studies on the immunochemistry of human fibrinopeptide B suggest that by restricting the size of the immunizing antigen, one can readily obtain antibodies which are relatively. restricted as determined by isoelectric focusing experiments, and which also offer great specificity. The potential for developing new antibody and T cell orobes into workable assays for detecting circulating fibrin(ogen) derivatives remains to be exolored.

scholarly journals Development of NCOVID-19 Colloidal Gold Immunochromatographic Test Strip

2021 ◽  
Vol 10 (1) ◽  
pp. 1
Author(s):  
Wenyi Liu ◽  
Zhaohua Li
Keyword(s):  
Colloidal Gold ◽  
Low Cost ◽  
Virus Detection ◽  
High Specificity ◽  
Accurate Method ◽  
Rapid Screening ◽  
Nucleic Acid Detection ◽  
Specificity And Sensitivity ◽  
Control Serum

<div><p>Purpose: To establish a fast, simple and accurate method and immunoassay test card for the detection of new coronavirus (nCOVID-19) antigen. Methods: In this study, colloidal gold immunochromatography technology was used to detect nCOVID-19 virus antigens through the sandwich method. At the same time, the preparation plan of colloidal gold was improved, and the application of rapid immune-diagnosis technology in other fields was developed. In this study, purified recombinant nCOVID-19 nucleocapsid protein is used as the antigen to prepare murine monoclonal antibodies. The BN02 antibody produced by the mouse is used as the detection antibody to couple with colloidal gold, forming a gold-labeled complex probe. BN9m1 is used as the coating antibody for the C-line, and ProA is used for the T-line. The polymerization of colloidal gold particles enables us to detect the new coronavirus antigen’s appearance. Thus an in vitro rapid detection kit for virus detection can be made. Results: The positive detection rate of the antigen quality control serum with this colloidal gold reagent was 100%. The specificity was 100%, and the sensitivity was 1ng/ml.  Conclusion: The nCOVID-19 antigen detection reagent (colloidal gold method) developed in this research has high specificity and sensitivity, and can be used in conjunction with nucleic acid detection. As a means of detecting nCOVID-19, it can achieve qualitative and rapid screening of samples with advantage such as accuracy, repeatability, and low cost.</p></div>

Sensitive Radioimmunoassay for Detection of O6-Ethyldeoxyguanosine in D N A Exposed to the Carcinogen Ethylnitrosourea in vivo or in vitro

1978 ◽  
Vol 33 (11-12) ◽  
pp. 897-901 ◽  
Author(s):  
Rolf Müller ◽  
Manfred F Rajewsky
Keyword(s):  
Nucleic Acid ◽  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Association Constant ◽  
High Specificity ◽  
Diploid Cell ◽  
Molar Ratio

O8-ethyl-2′-deoxyguanosine (O8-EtdGuo) is a major premutational product formed in both in­tracellular DNA and in purified DNA in vitro, after exposure to the potent alkylating carcinogen N-ethyl-N-nitrosourea (EtNU). Antibodies directed against O8-EtdGuo were obtained by immunizing rabbits with a conjungate of O6-EtGuo and bovine serum albumin. In a competitive radioimmuno­assay (RIA), with O8-Et[8,5′·3H]dGuo as a tracer and various alkylated and natural nucleic acid components as inhibitors, these antibodies show high specificity for O6-EtdGuo and detect this product at a level of 0.3 picomol (antibody association constant, 7×108l/mol). In a sample of 130 μg of hydrolyzed DNA, O6-EtdGuo can thus be measured at a molar ratio of O8-EtdGuo/2′- deoxyguanosine of about 3×10-8, i. e., about 5×103 O8-EtdGuo molecules per diploid cell. Exam­ples are given for the quantitation of Oe-EtdGuo in DNA exposed to EtNU in vivo or in vitro.

A Physiologic Regulator of Collagen-Induced Platelet Aggregation: Inhibition by Clq

1981 ◽  
Vol 45 (02) ◽  
pp. 110-115 ◽  
Author(s):  
György Csákó ◽  
Eva A Suba
Keyword(s):  
Platelet Aggregation ◽  
Human Platelet ◽  
Platelet Reactivity ◽  
High Specificity ◽  
Turbidimetric Method ◽  
Specific Manner ◽  
Aggregation Inhibition ◽  
Different Tissues

SummaryPlatelet aggregations were studied by a turbidimetric method in citrated human platelet-rich plasmas (PRP) in vitro. Human Clq inhibited the aggregations caused by collagens derived from different tissues and species. Clq was needed by weight in comparable quantities to collagen for neutralizing the aggregating effect. The dependence of the inhibitory reaction on the preincubation of platelets with Clq and the differences in the occurrence of aggregating substances in supernatants of PRP triggered with collagen in the presence or absence of Clq, confirmed that Clq exerts its effect by preventing fixation of collagen to platelets. In addition, the high specificity of the inhibitory action of Clq for collagen-induced platelet aggregation was demonstrated by results obtained for testing a variety of aggregating agents in combination with Clq and/or collagen.Since normal concentrations of Clq in the blood are in the range of inhibitory doses of Clq for collagen-induced platelet aggregations in vitro and upon activation of complement Clq is known to dissociate from Cl, it is proposed that Clq may participate in a highly specific manner in regulating platelet reactivity to collagen in vivo.

Prospective Application of Aptamer-based Assays and Therapeutics in Bloodstream Infections

2020 ◽  
Vol 20 (10) ◽  
pp. 831-840
Author(s):  
Weibin Li
Keyword(s):  
Pathogenic Bacteria ◽  
Genetic Diagnosis ◽  
Bloodstream Infections ◽  
High Specificity ◽  
Peptide Sequence ◽  
High Morbidity ◽  
Specificity And Sensitivity ◽  
Prospective Application ◽  
Small Molecule Therapeutics

Sepsis is still a severe health problem worldwide with high morbidity and mortality. Blood bacterial culture remains the gold standard for the detection of pathogenic bacteria in bloodstream infections, but it is time-consuming, and both the sophisticated equipment and well-trained personnel are required. Immunoassays and genetic diagnosis are expensive and limited to specificity and sensitivity. Aptamers are single-stranded deoxyribonucleic acid (ssDNA) and ribonucleic acid (RNA) oligonucleotide or peptide sequence generated in vitro based on the binding affinity of aptamer-target by a process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX). By taking several advantages over monoclonal antibodies and other conventional small-molecule therapeutics, such as high specificity and affinity, negligible batch-to-batch variation, flexible modification and production, thermal stability, low immunogenicity and lack of toxicity, aptamers are presently becoming promising novel diagnostic and therapeutic agents. This review describes the prospective application of aptamerbased laboratory diagnostic assays and therapeutics for pathogenic bacteria and toxins in bloodstream infections.

IMMU-53. STING-ING GLIOBLASTOMA WITH SPHERICAL NUCLEIC ACIDS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi104-vi105
Author(s):  
Akanksha Mahajan ◽  
Lisa Hurley ◽  
Serena Tommasini-Ghelfi ◽  
Corey Dussold ◽  
Alexander Stegh ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
High Surface ◽  
Biological Properties ◽  
Innate Immune ◽  
Limiting Factors ◽  
Nucleic Acid Delivery ◽  
Sting Pathway ◽  
Spherical Nucleic Acids

Abstract The Stimulator of Interferon Genes (STING) pathway represents a major innate immune sensing mechanism for tumor-derived DNA. Modified cyclic dinucleotides (CDNs) that mimic the endogenous STING ligand cGAMP are currently being explored in patients with solid tumors that are amenable to intratumoral delivery. Inadequate bioavailability and insufficient lipophilicity are limiting factors for clinical CDN development, in particular when consideration is given to systemic administration approaches. We have shown that the formulation of oligonucleotides into Spherical Nucleic Acid (SNA) nanostructures, i.e.,the presentation of oligonucleotides at high density on the surface of nanoparticle cores, lead to biochemical and biological properties that are radically different from those of linear oligonucleotides. First-generation brain-penetrant siRNA-based SNAs (NCT03020017, recurrent GBM) have recently completed early clinical trials. Here, we report the development of a STING-agonistic immunotherapy by targeting cGAS, the sensor of cytosolic dsDNA upstream of STING, with SNAs presenting dsDNA at high surface density. The strategy of using SNAs exploits the ability of cGAS to raise STING responses by delivering dsDNA and inducing the catalytic production of endogenous CDNs. SNA nanostructures carrying a 45bp IFN-simulating dsDNA oligonucleotide, the most commonly used and widely characterized cGAS activator, potently activated the cGAS-STING pathway in vitro and in vivo. In a poorly immunogenic and highly aggressive syngeneic mouse glioma model, in which tumours were well-established, only one dose of intranasal treatment with STING-SNAs decelerated tumour growth, improved survival and importantly, was well-tolerated. Our use of SNAs addresses the challenges of nucleic acid delivery to intracranial tumor sites via intranasal route, exploits the binding of dsDNA molecules on the SNA surface to enhance the formation of a dimeric cGAS:DNA complex and establishes cGAS-agonistic SNAs as a novel class of immune-stimulatory modalities for triggering innate immune responses against tumor.

Framework nucleic acid programmed combinatorial delivery nanocarriers for parallel and multiplexed analysis

2021 ◽  
Author(s):  
Yinghui Feng ◽  
Qi Liu ◽  
Miao Chen ◽  
Xinyi Zhao ◽  
Lumin Wang ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Multiplexed Analysis

Herein we report a framework nucleic acid programmed strategy to develop nanocarriers to precisely and independently package multiple homo- and heterogeneous cargos in vitro and in vivo, thereby enabling multiplexed...

scholarly journals A Novel Panel of Rabbit Monoclonal Antibodies and Their Diverse Applications Including Inhibition of Clostridium perfringens Epsilon Toxin Oligomerization

Antibodies ◽  
2018 ◽  
Vol 7 (4) ◽  
pp. 37 ◽  
Author(s):  
Jennifer Linden ◽  
Kiel Telesford ◽  
Samantha Shetty ◽  
Paige Winokour ◽  
Sylvia Haigh ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Monoclonal Antibodies ◽  
Western Blot ◽  
Clostridium Perfringens ◽  
Specificity And Sensitivity ◽  
Epsilon Toxin ◽  
Blocking Antibodies

The pore-forming epsilon toxin (ETX) produced by Clostridium perfringens is among the most lethal bacterial toxins known. Sensitive antibody-based reagents are needed to detect toxin, distinguish mechanisms of cell death, and prevent ETX toxicity. Using B-cell immuno-panning and cloning techniques, seven ETX-specific monoclonal antibodies were generated from immunized rabbits. ETX specificity and sensitivity were evaluated via western blot, ELISA, immunocytochemistry (ICC), and flow cytometry. ETX-neutralizing function was evaluated both in vitro and in vivo. All antibodies recognized both purified ETX and epsilon protoxin via western blot with two capable of detecting the ETX-oligomer complex. Four antibodies detected ETX via ELISA and three detected ETX bound to cells via ICC or flow cytometry. Several antibodies prevented ETX-induced cell death by either preventing ETX binding or by blocking ETX oligomerization. Antibodies that blocked ETX oligomerization inhibited ETX endocytosis and cellular vacuolation. Importantly, one of the oligomerization-blocking antibodies was able to protect against ETX-induced death post-ETX exposure in vitro and in vivo. Here we describe the production of a panel of rabbit monoclonal anti-ETX antibodies and their use in various biological assays. Antibodies possessing differential specificity to ETX in particular conformations will aid in the mechanistic studies of ETX cytotoxicity, while those with ETX-neutralizing function may be useful in preventing ETX-mediated mortality.

Sign in / Sign up

Export Citation Format

Share Document