Colorimetric detection of DNA sequences based on electrostatic interactions with unmodified gold nanoparticles

We find that single- and double-stranded oligonucleotides have different propensities to adsorb on gold nanoparticles in colloidal solution. We use this observation to design a hybridization assay based on color changes associated with gold aggregation. Because the underlying adsorption mechanism is electrostatic, no covalent functionalization of the gold, the probe, or the target DNA is required. Hybridization conditions can be optimized because it is completely separated from the detection step. The assay is complete within 5 min, and <100 femtomoles of target produces color changes observable without instrumentation. Single-base-pair mismatches are easily detected.     

Hybridoma antibody-based competitive ELISA in Schistosoma japonicum infection

A competitive enzyme-linked immunosorbent assay (ELISA) has been developed and compared with the circumoval precipitin test (COPT) for diagnosis of schistosomiasis japonica using Philippine sera. The assay is based on the inhibition, by sera, of the binding of a penicillinase-conjugated hybridoma-derived antibody, I. 134, to a crude Schistosoma japonicum adult worm extract. A change in pH subsequent to addition of the substrate is used as the indicator system. Development of the color change in this assay is relatively slow, a fact which presumably facilitates detection of inhibition by serum. Relative to the COPT, no false positive reactions were obtained and the false negative rate was less than 10%. A wide range of inhibitory titers was obtained using sera in the competitive ELISA similar to that found in a competitive radioimmunoassay using 125I-labeled I. 134. The competitive ELISA will be of more general application for diagnosis of schistosomiasis japonica than the competitive RIA using hybridoma antibodies, and will provide more precise quantitative information than is obtainable in the COPT.     

Rapid detection of KPC-producing Enterobacterales by using a modified Carba NP test with imipenem/relebactam

IntroductionHere, we propose a novel modified Carba NP test for detecting KPC-producing Enterobacterales using imipenem/relebactam.Material and methodsThe test performance was evaluated in a random selection of 160 previously molecularly characterized clinical isolates carrying the 110 blaKPC, 1 bla_GES, 12 bla_VIM, 4 bla_IMP, 3 bla_NDM and 42 bla_OXA-48-like genes. The proposed method relies on the detection of imipenem hydrolysis in an imipenem/relebactam antibiotic solution and subsequent visual interpretation by color change.ResultsAll class A producing Enterobacterales (111/111) were detected using imipenem/relebactam as no visual appreciation of color change was perceived due to a nule hydrolysis of imipenem in the antibiotic solution. Overall, the assay showed 100% sensitivity (111/111) and specificity (69/69) for detecting class A KPC-producing Enterobacterales.DiscussionThe biochemical assay provides very reliable results for detecting KPC-producing Enterobacterales, with a turnaround time of less than 1 hour, minimum handling and no specialized equipment required.     

A simple Escherichia coli system for monitoring HIV protease activity: analysis of two temperature-sensitive protease mutants

A simple Escherichia coli system has been developed for the detection of human immunodeficiency virus (HIV) protease activity. In this system, the protease sequence is placed downstream of the HIV gag polypeptide in an operon arrangement. Upon expression of the operon, gag serves as the substrate for the protease; the level of protease activity can be determined by measurement of the cleavage product of gag in cell extracts by Western immunoblotting. This system is useful in both detection of protease mutations generated by mutagenesis and in testing substrate specificity of the protease by mutagenesis of the gag sequence. Using this system, we have observed that modification of the N-terminus of HIV protease renders the enzyme temperature sensitive; the temperature sensitivity is made more pronounced by the conserved change of valine to isoleucine at residue eleven.     

A protease responsible for post-translational cleavage of a conserved Asn-Gly linkage in glycinin, the major seed storage protein of soybean

The assembly of 11S globulin seed storage proteins in plants is regulated in part by the activity of a protease that cleaves between asparagine and glycine residues. Post-translational cleavage of subunit precursors into acidic and basic polypeptides is associated with the ability of subunits in trimers to aggregate into hexamers in vitro. An activity is present in extracts from immature soybean seeds that specifically cleaves immature 11S seed storage proteins of soybean and Vicia faba into the polypeptides of the mature proteins. Sequence microanalysis has been used to demonstrate that proglycinin and prolegumin are cut at the legitimate site when proteins synthesized in vitro are used as substrates. A single amino acid change in the cleavage site renders the substrate uncleavable. The protease responsible for this activity also hydrolyzes a synthetic octapeptide whose sequence reproduces four amino acids on either side of the glycinin subunit G4 cleavage site. This assay permitted the purification and characterization of the protease. It is a glycosylated enzyme with an acidic pH optimum and a molecular mass of about 45 kDa in solution.     

Development of a Bio-Layer Interferometry-Based Protease Assay Using HIV-1 Protease as a Model

Proteolytic enzymes have great significance in medicine and the pharmaceutical industry and are applied in multiple fields of life sciences. Therefore, cost-efficient, reliable and sensitive real-time monitoring methods are highly desirable to measure protease activity. In this paper, we describe the development of a new experimental approach for investigation of proteolytic enzymes. The method was designed by the combination of recombinant fusion protein substrates and bio-layer interferometry (BLI). The protease (PR) of human immunodeficiency virus type 1 (HIV-1) was applied as model enzyme to set up and test the method. The principle of the assay is that the recombinant protein substrates immobilized to the surface of biosensor are specifically cleaved by the PR, and the substrate processing can be followed by measuring change in the layer thickness by optical measurement. We successfully used this method to detect the HIV-1 PR activity in real time, and the initial rate of the signal decrease was found to be proportional to the enzyme activity. Substrates representing wild-type and modified cleavage sites were designed to study HIV-1 PR’s specificity, and the BLI-based measurements showed differential cleavage efficiency of the substrates, which was proven by enzyme kinetic measurements. We applied this BLI-based assay to experimentally confirm the existence of extended binding sites at the surface of HIV-1 PR. We found the measurements may be performed using lysates of cells expressing the fusion protein, without primary purification of the substrate. The designed BLI-based protease assay is high-throughput-compatible and enables real-time and small-volume measurements, thus providing a new and versatile approach to study proteolytic enzymes.     

Peptide inhibitors of HIV-1 protease and viral infection of peripheral blood lymphocytes based on HIV-1 Vif

We recently reported that HIV-1 Vif (virion infectivity factor) inhibits HIV-1 protease in vitro and in bacteria, suggesting that it may serve as the basis for the design of new protease inhibitors and treatment for HIV-1 infection. To evaluate this possibility, we synthesized peptide derivatives from the region of Vif, which inhibits protease, and tested their activity on protease. In an assay of cleavage of virion-like particles composed of HIV-1 Gag precursor polyprotein, full-length recombinant Vif, and a peptide consisting of residues 21–65 of Vif, but not a control peptide or BSA, inhibited protease activity. Vif_21–65 blocked protease at a molar ratio of two to one. We then tested this peptide and a smaller peptide, Vif_41–65, for their effects on HIV-1 infection of peripheral blood lymphocytes. Both Vif peptides inhibited virus expression below the limit of detection, but control peptides had no effect. To investigate its site of action, Vif_21–65 was tested for its effect on Gag cleavage by protease during HIV-1 infection. We found that commensurate with its reduction of virus expression, Vif_21–65 inhibited the cleavage of the polyprotein p55 to mature p24. These results are similar to those obtained by using Ro 31–8959, a protease inhibitor in clinical use. We conclude that Vif-derived peptides inhibit protease during HIV-1 infection and may be useful for the development of new protease inhibitors.     

A Gold Nanoparticle and Aflatoxin B1-BSA Conjugates Based Lateral Flow Assay Method for the Analysis of Aflatoxin B1

A rapid and simple immuno-chromatographic assay was developed to detect aflatoxin B1 (AFB1). The assay was based on a modified competitive binding format using colloidal gold and polyclonal antibody (Pab) conjugates. The anti-AFB1 Pab was immobilized to a defined detection zone on a porous nitrocellulose membrane and colloidal gold particles were conjugated to AFB1-BSA which served as a detection reagent. The AFB1-containing sample was added to the membrane and allowed to move with AFB1-BSA-coated particles dried on the conjugation pad. The mixture was then passed along the porous membrane by capillary action past the Pab in the detection zone, which captured AFB1 or AFB1-BSA. AFB1 in the sample inhibits binding of AFB1-BSA conjugated gold particles to the Pab and prevents formation of a red color dot. In the absence of AFB1, AFB1-BSA conjugated gold particles bound to the Pab, give a red color within this detection zone. With this method, 10 μg/mL of AFB1 was detected in less than 10 min. The developed AFB1 assay also showed no cross reaction to Ochratoxin A (OTA).     

多交叉置换扩增技术在单增李斯特菌检测中的应用及评价

目的 应用多交叉置换扩增(MCDA)技术建立单增李斯特菌的简单、快速、敏感且特异的诊断方法, 并对该方法进行敏感度、特异性及实际应用评价。方法 根据单增李斯特菌的种特异性基因lmo0733设计引物,采用荧光染料颜色变化、实时浊度检测及琼脂糖凝胶电泳三种方法确认MCDA产物,对MCDA引物进行最佳反应条件、敏感度、特异性评估,并对实际样本进行检测。结果 单增李斯特菌MCDA检测方法的最佳反应温度为61 ℃。单增李斯特菌MCDA检测方法的敏感度为10 fg/反应,分别是环介导等温扩增(LAMP)技术和交叉引物恒温扩增(CPA)技术的25倍和250倍。对153份鼠粪便标本2次增菌培养物的检测结果证实,本研究建立的单增李斯特菌MCDA检测方法的检测能力与分离培养方法(ISO 11290-1)相同,且优于LAMP、CPA和PCR方法。结论 MCDA方法作为一种快速、敏感和高效的检测方法可以应用于食品行业及临床标本中单增李斯特菌的检测。     

A protease assay for two-photon crosscorrelation and FRET analysis based solely on fluorescent proteins

GFP and the red fluorescent protein, DsRed, have been combined to design a protease assay that allows not only for fluorescence resonance energy transfer (FRET) studies but also for dual-color crosscorrelation analysis, a single-molecule-based method that selectively probes the concomitant movement of two distinct tags. The measurement principle is based on a spectrally resolved detection of single molecules diffusing in and out of a diffraction-limited laser focus. Double-labeled substrate molecules are separated into two single-labeled products by specific cleavage at a protease cleavage site between the two flanking tags, DsRed and GFP, thus disrupting joint fluctuations in the two detection channels and terminating FRET between the two labels. In contrast to enzyme assays based solely on FRET, this method of dual-color crosscorrelation is not limited to a certain range of distances between the fluorophores and is much more versatile with respect to possible substrate design. To simplify the measurement setup, two-photon excitation was used, allowing for simultaneous excitation of both tags with a single infrared laser wavelength. The general concept was experimentally verified with a GFP-peptide-DsRed construct containing the cleavage site for tobacco etch virus protease. Two-photon excitation in the infrared and the use of cloneable tags make this assay easily adaptable to intracellular applications. Moreover, the combination of FRET and crosscorrelation analysis in a single-molecule-based approach promises exciting perspectives for miniaturized high-throughput screening based on fluorescence spectroscopy.     

CLINICAL SIGNIFICANCE OF ADAPTIVE INDEX OF HEMOGLOBIN COLOR ENHANCEMENT FOR ENDOSCOPIC DIAGNOSIS OF SUPERFICIAL TYPE COLORECTAL TUMORS

We examined the clinical significance of adaptive index of hemoglobin (IHb) color enhancement (color enhancement) for the detection of superficial type colorectal tumors. In this prospective study, the detection rate of superficial type tumors with colonoscopic examination was significantly higher in the color enhancement group than in the control group. The colonoscopic findings made more distinct by color enhancement, leading to tumor detection, were tumor redness and interrupted vascular networks around the tumor. These results demonstrate that color enhancement is effective for the detection and diagnosis of superficial type colorectal tumors.     

Communication between the active sites and dimer interface of a herpesvirus protease revealed by a transition-state inhibitor

Structurally diverse organophosphonate inhibitors targeting the active site of the enzyme were used to investigate the relationship of the active site and the dimer interface of wild-type protease in solution. Positional scanning synthetic combinatorial libraries revealed Kaposi's sarcoma-associated herpesvirus protease to be highly specific, even at sites distal to the peptide bond undergoing hydrolysis. Specificity results were used to synthesize a hexapeptide diphenylphosphonate inhibitor of Kaposi's sarcoma-associated herpesvirus protease. The transition state analog inhibitors covalently phosphonylate the active site serine, freezing the enzyme structure during catalysis. An NMR-based assay was developed to monitor the native monomer-dimer equilibrium in solution and was used to demonstrate the effect of protease inhibition on the quaternary structure of the enzyme. NMR, circular dichroism, and size exclusion chromatography analysis showed that active site inhibition strongly regulates the binding affinity of the monomer-dimer equilibrium at the spatially separate dimer interface of the protease, shifting the equilibrium to the dimeric form of the enzyme. Furthermore, inhibitor studies revealed that the catalytic cycles of the spatially separate active sites are independent. These results (i) provide direct evidence that peptide bond hydrolysis is integrally linked to the quaternary structure of the enzyme, (ii) establish a molecular mechanism of protease activation and stabilization during catalysis, and (iii) highlight potential implications of substoichiometric inhibition of the viral protease in developing herpesviral therapeutics.     

An investigation of non-specific reactions in measurement of plasma (1-->3)-beta-D-glucan

We investigated the detection of non-specific reaction in measurement of plasma (1-->3)-beta-D-glucan (beta-glucan) by alkaline treatment, chromogenic automated kinetic assay (alkaline-kinetic assay) and dilution and heating method, chromogenic endpoint assay (dilution heating endpoint assay). In this study, we reexamined the values of beta-glucan by both methods with and without 4-amidinophenyl benzoate hydrochloride (APB) as protease inhibitor that blocks Limulus reaction in the 142 serum samples from 142 patients who had been treated and measured beta-glucan in Kawasaki medical school hospital between January 1999 and May 1999. Non-specific reactions were judged by the calculated value under APB additive condition. The non-specific reactions were found in 135 of total 142 samples (95.1%) in the alkaline-kinetic assay while no non-specific reactions were recognized in dilution heating endpoint assay. The alkaline-kinetic assay has been used widely and been evaluated it's usefulness because of good sensitivity. However, we found very high frequency of nonspecific reaction in this method. Further studies are needed to define the reasons of non-specific reaction. On the other hand, although non-specific reactions were not detected in dilution heating end-point assay, it's clinical utilities should be evaluated in future clinical studies.     

Establishment of a simple assay in vitro for hepatitis C virus NS3 serine protease based on recombinant substrate and single—Chain protease

AIM: To establish a simple and convenient assay in vitro for the Hepatitis C virus NS3 serine protease based on the recombinant protease and substrate, and to evaluate its feasibility in screening the enzyme inhibitors. METHODS: Based on the crystallographic structure of hepatitis C virus (HCV) serine protease, a novel single-chain serine protease was designed, in which the central sequence of cofactor NS4A was linked to the N-terminus of NS3 serine protease domain via a flexible linker GSGS. The fusion gene was obtained by two-step PCR that was carried out with three primers and then cloned into the prokaryotic expression vector pQE30, and the recombinant clone was verified by DNA sequencing. The single-chain recombinant protease was expressed when the E.coli was induced with IPTG and the expression conditions were optimized to produce large amount of soluble protease. The recombinant substrate NS5ab that covers the cleavage point NS5A/B was also expressed in E.coli. Both of the protease and substrate were purified by using Ni-NTA agarose metal affinity resin, then they were mixed together in a specific buffer, and the mixture was analyzed by SDS-PAGE. The cleavage system was used to evaluate some compounds for their inhibitory activity on serine protease. RESULTS: The single-chain recombinant protease was over-expressed as soluble protein when the E.coli was induced at a low dosage of IPTG (0.2 mM) and cultured at a low temperature (15 degrees ). The protease was purified by using Ni-NTA agarose metal affinity resin (the purity is over 95 %). The recombinant substrate NS5ab was expressed in an insoluble form and could refold successfully after purification and dialysis. A simple and convenient assay in vitro was established, in which the purified single-chain serine protease could cleave the recombinant substrate NS5ab into two fragments that were visualized by SDS-PAGE. PMSF had an effect on inhibiting activity of serine protease, while EDTA had not. CONCLUSION: A simple and convenient assay in vitro for hepatitis C virus NS3 serine protease is based on recombinant substrate NS5ab and single-chain serine protease. This assay can be used in screening of enzyme inhibitors.     

Polymorphisms in p1-p6/p6* of HIV type 1 can delay protease autoprocessing and increase drug susceptibility

Maturation of infectious human immunodeficiency virus type 1 (HIV-1) particles requires proteolytic cleavage of structural polyproteins by viral protease. Inhibition of protease is a powerful tool for the treatment of HIV infection. Using a well-established phenotypic drug susceptibility assay, we found that sequences outside of the protease gene can modulate the susceptibility to protease inhibitors (PIs). Chimeric viruses carrying p1-p6/p6* sequences from patient isolates in the context of an NL4-3 molecular clone exhibited increased PI susceptibility. Furthermore, this phenotype was associated with a delay in protease autoprocessing in virions and a reduction in replication capacity. We propose that the interplay between protease and the C terminus of Gag is critical for proper protease activity and mismatches between these regions can reduce viral replication and increase drug susceptibility.     

The degradation of monoiodotyrosyl insulin isomers by insulin protease

The four single-site monoiodotyrosyl insulin isomers were synthesized by lactoperoxidase-catalyzed iodination of porcine insulin and were separated from one another by high performance liquid chromatography. The susceptibility of the four isomers (A14-, A19-, B16-, and B26-monoiodotyrosyl insulin) to degradation by purified insulin protease was examined using several different assay methods, including trichloroacetic acid precipitation, immunoprecipitation, and Sephadex G-50 chromatograpy. Using trichloroacetic acid precipitation, isomer susceptibility, determined from the initial rate of hydrolysis, was highest with the A14 isomer, lowest with the A19 isomer, and intermediate and roughly equal with the two B-chain-labeled isomers. Based upon the initial rate of isomer hydrolysis, the Michaelis Menten constant (Km) of insulin protease was higher for the B16 isomer (55 nM) than for the other three isomers, whose Km values were not different from one another (A14 = 24 nM; A19 = 35 nM; B26 = 29 nM). In addition, the values for maximum velocity (Vmax) were higher for the A14 and B26 isomers than for the A19 and B16 isomers. However, during incubation, the order of isomer susceptibility to insulin protease changed to B26 greater than A14 greater than A19 greater than B16. This change in apparent isomer susceptibility was prevented by including in the incubation mixture a rat renal peptidase, which did not degrade the intact isomers, suggesting that insulin protease converted the isomers to trichloroacetic acid-soluble products via trichloroacetic acid-precipitable intermediates. Using the immunoprecipitation assay, the susceptibility of isomers to hydrolysis did not change during incubation, but remained highest with the A14 isomer, lowest with the A19 isomer, and intermediate with the two B-chain-labeled isomers, of which the B16 isomer was degraded more rapidly. Each isomer was converted more rapidly to nonimmunoprecipitable products than to trichloroacetic acid-soluble products, implying that insulin protease converted the isomers to trichloroacetic acid-precipitable, nonimmunoprecipitable intermediates, which it then converted to trichloroacetic acid-soluble form. Using Sephadex G-50 chromatography (SGC) assay, the susceptibility of isomers to hydrolysis did not change during incubation, but remained highest with the A14 isomer, lowest with the A19 isomer, and intermediate with the two B-chain-labeled isomers, of which the B16 isomer was hydrolyzed more rapidly. With the exception of the A19 isomer, isomer hydrolysis appeared faster with SGC assay than with either of the other two assays.(ABSTRACT TRUNCATED AT 400 WORDS)     

Establishment of a cell-based assay system for hepatitis C virus serine protease and its primary applications

AIM: To establish an efficient, sensitive, cell-based assay system for NS3 serine protease in an effort to study further the property of hepatitis C virus (HCV) and develop new antiviral agents. METHODS: We constructed pCI-neo-NS3/4A-SEAP chimeric plasmid, in which the secreted alkaline phosphatase (SEAP) was fused in-frame to the downstream of NS4A/4B cleavage site. The protease activity of NS3 was reflected by the activity of SEAP in the culture media of transient or stable expression cells. Stably expressing cell lines were obtained by G418 selection. Pefabloc SC, a potent irreversible serine protease inhibitor, was used to treat the stably expressing cell lines to assess the system for screening NS3 inhibitors. To compare the activity of serine proteases from 1b and 1a, two chimeric clones were constructed and introduced into both transient and stable expression systems. RESULTS: The SEAP activity in the culture media could be detected in both transient and stable expression systems, and was apparently decreased after Pefabloc SC treatment. In both transient and stable systems, NS3/4A-SEAP chimeric gene from HCV genotype 1b produced higher SEAP activity in the culture media than that from 1a. CONCLUSION: The cell-based system is efficient and sensitive enough for detection and comparison of NS3 protease activity, and screening of anti-NS3 inhibitors. The functional difference between NS3/4A from 1a and 1b subtypes revealed by this system provides a clue for further investigations.     

Application of the enzyme linked immunospecific assay (ELISA) for the detection of platelet antibodies

Many methods have been described to identify platelet antibody, but they are either not very sensitive or too complex for general use. Therefore, we have developed an enzyme immunoassay for the detection of platelet antibodies in serum. The method involves incubating platelets with serum antibody; any attached antibody is shown by the addition of an enzyme (alkaline phosphatase) labeled anti-human IgG, followed by assay of the enzyme reaction with its substrate. The reaction product is indicated by a color change, which is proportional to the antibody concentration. Assay conditions such as the use of paraformaldehyde fixed versus unfixed platelets, conjugate dilutions, and substrate concentration and incubation time were investigated. Positive results were obtained in 16 of 19 sera of patients with various diseases including 2 of 4 patients with idiopathic thrombocytopenic purpura, 2 of 2 with post-transfusion purpura, 2 of 3 with neonatal purpura, and all 9 polytransfused patients. Sensitivity and specificity were 84% and 98%, respectively. Also, enzyme linked immunospecific assay (ELISA) was found to be superior to the lymphocytotoxicity (LCT) and platelet immunofluorescence test (PIIFT) for platelet antibody identification.