30 results about "Scatchard plot" patented technology

A method for determining a soluble human ST2 in a sample conveniently at a high sensitivity and an assay kit are provided. By an immunological method comprising a step for bringing a sample into contact with an immobilized antibody formed by binding to an insoluble support a first anti-human ST2 antibody which binds specifically to a non-denatured human ST2, a step for labelling a first reaction product generated in the previous step by reacting said first reaction product with a second anti-human ST2 antibody which binds specifically to a non-denatured human ST2 by recognizing a site different from the site on ST2 where said first anti-human ST2 antibody binds and which is labelled with a label, and a step for determining the amount of the label on said first reaction product which has been labelled, a soluble human ST2 in a sample is determined. In addition, a recombinant ST2 is employed as a standard to prepare a calibration curve, based on which the ST2 in a sample is quantified.

The present invention provides a method and apparatus for detecting the noncovalent binding of a potential ligand (such as a drug candidate) to a target, e.g. a biochemical macromolecule such as a protein. The method is based on the Taylor dispersion of an initially sharp boundary between a carrier solution, and an analyte solution that contains the potential ligand(s) and the target. Dispersion profiles of one or more potential ligands are monitored by mass spectrometry at the exit of the laminar flow tube. Potential ligands will usually be relatively small molecules that have large diffusion coefficients. In the absence of any noncovalent interactions in solution, very steep dispersion profiles are expected for these potential ligands. However, a ligand that binds to a large target in solution, will show an apparent diffusion coefficient that is significantly reduced, thus resulting in a more extended dispersion profile. Noncovalent binding can therefore be detected by monitoring dispersion profiles of potential ligands in the presence and in the absence of the target. In contrast to other mass spectrometry-based methods for detecting noncovalent interactions, this method does not rely on the preservation of specific noncovalent interactions in the gas phase. This method has an excellent sensitivity and selectivity, therefore it can be used for testing multiple potential ligands simultaneously. The method is therefore useful for the high throughput screening of compound libraries.

The invention provides a method for determining the receptor affinity of a GLP-1 receptor agonist. The method is based on a tool cell line, namely CHO-GLP-1R-CRE-Luc<+>, when a sample to be determined is combined with GLP-1 receptor on the surface of a cell, the receptor-mediated signal cascade reaction can be activated, cAMP-response element (CRE) is activated specifically, the expression of the luciferase reporter gene is promoted, the quantity of luciferase in cytosol is detected for drawing and fitting to obtain a dose-effect curve of acting of the sample and the GLP-1 receptor; and the half effective concentration (EC50) is calculated. By inspecting and optimizing all influence factors in the determining process, the method for determining the receptor affinity of the GLP-1 receptor agonist is finally established. The method has the advantages of being high in specificity, precision and accuracy, good in durability and convenient to operate, and the like. The method can be used for determining the receptor affinity of the GLP-1 receptor agonist, and thus such type of drug can be fast evaluated and screened.

The invention discloses a bispecific antibody biological activity and titer detection method. The method includes: detecting two antigen binding sites of a bispecific antibody at the same time; performing four-parameter fitting according to a fluorescence ratio and the concentration of a standard sample to obtain a standard curve; calculating the concentration of a to-be-tested sample according to the standard curve. By adoption of the method, intact antibody modules and cell strains high in bispecific binding activity and expression can be screened out more effectively; the method has the advantage of low reaction volume, and experiment reagent cost can be saved maximally; the method can be widely popularized and applied to CLD clone high-throughput screening.

The invention relates to the cloning of a transforming growth factor-beta (TGF-beta) superfamily type I receptor gene Tgfbrl1 of chlamys farreri in a molecular genetic marker technology, a screening and typing technology of a single nucleotide polymorphism (SNP) locus which is relevant with the weight of adductor muscles in the gene and a method for the application of the gene to the breeding of the high-yield chlamys farreri. A total-length complementary deoxyribonucleic acid (cDNA) sequence of the Tgfbr1 gene of the chlamys farreri is obtained by utilizing a homology-based cloning strategy; an SNP lotus is discovered by blastn comparison, and primers and a probe are designed for the locus; and SNP typing is performed in a natural population of the chlamys farreri by using a high-resolution melting curve technology, and the weight of the adductor muscle of an individual is measured. Statistic analysis displays that the loci c.1815C>T are obviously relevant with the weight of the adductor muscles of the chlamys farreri, and the weight of the adductor muscles of individuals with TT genetypes is obviously higher than that of the adductor muscles of individuals with CC and CT genetypes. In the selective breeding process of the high-yield chlamys farreri, the breeding candidate colonies of the chlamys farreri can be subjected to c.1815C>T typing, and the individuals of which the loci c.1815C>T are TT genetypes are used as a breeding parent preferably by combining typing information of other loci which are relevant with growth properties.

A method and apparatus for assaying a drug candidate with a biosensor having one or more sensing surface-bound biomolecules associated therewith are disclosed. The method comprises the steps of measuring the binding interaction between the drug candidate and the one or more sensing surface-bound biomolecules of the biosensor to obtain an estimate of at least one binding interaction parameter of the drug candidate, and then comparing the estimated binding interaction parameter against a mathematical expression correlated from binding interaction data associated with known drug compounds to determine an estimate of at least pharmacokinetic parameter of absorption, distribution, metabolism, or excretion (ADME) that is related to the drug candidate. The present invention allows for the simultaneous measurement of different pharmacokinetic parameters of the drug candidate, as well as an indication of the drug candidate's solubility, by use of a single analytical instrument. The pharmacokinetic data may be represented as a ADME characterization profile; such ADME profiles are of great utility for purposes of drug screening and lead optimization.

The invention provides a novel integrated structure and system-based approach for drug target prediction that enables the large-scale discovery of new targets for existing drugs Novel computer-readable storage media and computer systems are also provided. Methods and systems of the invention use novel sequence order-independent structure alignment, hierarchical clustering, and probabilistic sequence similarity techniques to construct a probabilistic pocket ensemble (PPE) that captures even promiscuous structural features of different binding sites for a drug on known targets. The drug's PPE is combined with an approximation of the drug delivery profile to facilitate large-scale prediction of novel drug-protein interactions with several applications to biological research and drug development.

Presented herein are methods, devices and kits for the mass spectrometric immunoassay (MSIA) of proteins present in complex biological fluids or extracts. Pipettor tips containing porous solid supports that are covalently derivatized with affinity ligand and used to extract specific proteins and their variants from various biological fluids. Nonspecifically bound compounds are rinsed from the extraction devices using a series of buffer and water rinses, after which the wild type protein (and / or its variants) are eluted directly onto a target in preparation for analysis such as matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). Mass spectrometry of the eluted sample then follows with the retained proteins identified via accurate molecular mass determination. Protein and variant levels can be determined using quantitative methods in which the protein / variant signals are normalized to signals of internal reference standard species (either doped into the samples prior to the MSIA analysis, or other endogenous protein co-extracted with the target proteins) and the values compared to a working curves constructed from samples containing known concentrations of the protein or variants. Such MSIA devices, kits and methods have significant application in the fields of; basic research and development, proteomics, protein structural characterization, drug discovery, drug-target discovery, therapeutic monitoring, clinical monitoring and diagnostics, as well as in the high throughput screening of large populations to establish and recognize protein / variant patterns that are able to differentiate healthy from diseased states.

The invention discloses a fluorescent probe for detecting ochratoxin A and a preparation method thereof, and belongs to the technical field of biochemistry. The fluorescent probe is characterized in that OTA is taken as a target, and a phage surface random display 12-mer peptide library is used to screen out a recombinant phage specifically bound to the OTA. By extracting single-stranded DNA of the recombinant phage for sequencing and sequence comparison, an OTA specific affinity ligand sequence, Met-Pro-Met-Phe-Lys-His-Arg-Met-Phe-His-Thr-His is obtained. Afterwards the fluorescent probe specifically bound to OTA, FITC-Acp-Met-Pro-Met-Phe-Lys-His-Arg-Met-Phe-His-Thr-His is obtained by solid-phase polypeptide synthesis and fluorescence labeling. The construction method comprises the following steps: 1, screening a specific affinity ligand; 2, extracting recombinant phage DNA and determining a target sequence; 3, preparing a fluorescent probe; and 4, establishing a standard detection curve. The fluorescent probe of the present invention has the beneficial effects of 1, replacing an OTA monoclonal antibody; and 2, qualitatively identifying OTA and detecting content of the OTA in a sample.

The invention provides a method for determining the binding amount of a biotinylated antibody of streptavidin labeled microspheres. The method comprises the following steps: 1) preparation of the biotinylated antibody: diluting 2mg of the antibody to 5mg / ml by using a PBS buffer solution, adding a certain amount of sulfo-NHS-LC-Biotin, uniformly shaking, reacting for 1 hour at room temperature, and dialyzing and purifying; 2) preparation of an acridinium ester-biotinylated antibody: diluting the biotinylated antibody in the step 1) to 2mg / ml, adding a certain amount of DMSO solution of acridinium ester, uniformly shaking, reacting for 2 hours in a dark place at room temperature, dialyzing and purifying in a dark place, and quantifying by using a BCA method; 3) drawing a standard curve, and reading a luminescence value RLU; and drawing an acridinium ester-biotinylated antibody addition mass-RLU relation curve. The method has the advantages that compared with a fluorescence method, the chemiluminescence method is higher in sensitivity and higher in anti-interference capability; and compared with a microsphere compound test method, the chemiluminescence method is simpler in operation and more accurate in result.

The invention discloses a method for detecting the concentration of a target molecule in a mixed system and a kit. The invention relates to a method for quantitatively detecting the concentration of a target molecule X. The method comprises the steps of adding a reporter molecule A into a reaction system containing the target molecule X for at least first given time, wherein specific binding activity exists between the reporter molecule A and the target molecule X; adding a competitive molecule B into the reaction system, wherein specific binding activity exists between the competitive molecule B and the reporter molecule A, the competitive molecule B and the target molecule X do not have any binding activity, and when the target molecule X does not exist, fluorescence intensity L0 with given intensity is emitted when the competitive molecule B is combined with the reporter molecule A; and uniformly mixing the system, and detecting the fluorescence intensity L of the system after waiting for at least a second given time, wherein in the ideal concentration range, a linear relation between the concentration of the target molecule X and the L0-L value can be established, and the established linear relation is used as a standard curve, and the concentration of the target molecule X can be calculated according to the actually measured fluorescence intensity change value L0-L. The invention also relates to a kit matched with the method.

The invention relates to the technical field of medical detection, in particular to a kit for detecting homocysteine. The kit for detecting homocysteine comprises a shell, a reagent and an informationcard, wherein a standard curve of absorbance values to concentration values is stored in the information card, a correction factor is stored in the information card, and the correction factor is usedfor correcting the concentration of homocysteine in a blood sample when the to-be-detected blood sample is whole blood; and the reagent comprises a treatment solution, a first reaction solution and asecond reaction solution, the treatment solution is used for converting protein-binding homocysteine in the blood sample into free homocysteine, and the first reaction solution and the second reaction solution are used for reacting with the free homocysteine. When the kit for detecting the homocysteine is in use, an analyzer can be used for directly obtaining the concentration of the homocysteineaccording to the absorbance value-to-concentration value standard curve which is stored in the information card, so that the kit is very convenient. In addition, the concentration of the homocysteinein the whole blood sample can be corrected through the correction factor.

The invention belongs to the field of molecular biological detection. In particular, the invention relates to the detection of SARS-CoV-2; more specifically, the invention relates to a composition, a kit and a method for detecting SARS-CoV-2 and application thereof. The invention provides a kit containing the composition, application of the composition and a method for detecting and typing the SARS-CoV-2 variant. By detecting variation sites with different characteristic functions, the Armike and Delta variants are subjected to typing, so that the typing detection of the SARS-CoV-2 variants is realized in a single-tube reaction system at the same time. Different strains can be treated differently, so that treatment and prevention are more efficient. The composition disclosed by the invention is low in cost, high in flux and simple and convenient to operate, and a result reading process can be judged through an amplification curve Ct value. The whole detection process is carried out under a single-tube closed condition, so that false positive and environmental pollution are avoided.

The invention discloses a method for measuring the dynamic loading capacity of an affinity chromatography filler, which comprises the following steps: 1) loading a sample onto a chromatographic column filled with the affinity chromatography filler to be screened, collecting flow-through liquid, and recording the loading volume; the sample loading volume is the total sample loading volume when the concentration of the penetrating protein sample corresponding to the ultraviolet absorption value reaches 5%-10% of the concentration of the target protein in the sample; the sample is a protein solution; and 2) calculating the dynamic loading capacity of the affinity chromatography filler according to the sample concentration-ultraviolet absorption value standard curve and the sample loading volume in the step 1). According to the property characteristics of affinity adsorption of the affinity filler and the antibody, an affinity adsorption kinetic model is constructed, the advantages of a traditional static binding capacity determination method and a current standard dynamic binding capacity determination method can be considered, and the method is short in time consumption, low in cost and high in accuracy.

The invention provides a ratio fluorescence immunochromatography detection test strip. The test strip comprises a sample pad, a marking pad, a coating film and a water absorption pad which are connected in sequence; a calibration line C, a test line T1 and a test line T2 are sequentially arranged on the coating film, and the line C is coated with an independent quality control antibody; the marking pad contains a fluorescently-labeled recombinant antigen and a fluorescently-labeled independent quality control antigen; the T1 line is coated with a recombinant antigen; and the T2 line is coated with a recombinant antigen ligand. According to the invention, the ratio (T1 / T2) of the fluorescence detection signals on the T1 line and the T2 line is determined, the ratio (T1 / T2) is positively correlated with the antibody concentration in the sample, and a correlation calibration curve equation is established so as to establish a qualitative or quantitative detection fluorescence immunochromatography detection method; according to the test strip and the method provided by the invention, the interference of a fluorescence detection background signal is overcome, and the sensitivity, the accuracy and the credibility of fluorescence immunochromatography are improved.

The invention discloses a quantitative determination method for a specific IgG antibody in plasma, which belongs to the field of antibody detection. The method comprises the following steps of 1) dividing one plasma sample into two groups as to-be-detected plasma and to-be-treated contrast plasma, (2) incubating excessive antigens and to-be-treated contrast plasma, and neutralizing to-be-detected antibodies in the to-be-treated contrast plasma to obtain contrast plasma, 3) taking to-be-detected plasma and contrast plasma as samples, combining the to-be-detected plasma and the contrast plasma with corresponding antigens connected to the solid-phase carrier, successively combining the antigen-antibody compound with IgG antibodies and signal molecules to generate quantifiable data, and subtracting two groups of quantifiable data to obtain detection data, 4) diluting thousands of mixed plasma serving as a standard substance into different concentrations according to the steps 1)-3) to make a standard curve, and 5) substituting detection data of a to-be-detected sample into the standard curve to obtain the content of the specific IgG antibody in the plasma. According to the determination method, the influence of non-specific binding can be eliminated, and the detection result is reliable.

The invention discloses a method for measuring kinetic and thermodynamic parameters on a solid-liquid interface, which comprises the steps of according to frequency-time curve graphs corresponding to various ligand solutions with different concentrations to be measured and frequency-time curve graphs of another ligand solution with the concentration to be measured at two different temperatures, analyzing and calculating an output frequency-time curve of a micro-cantilever sensor to obtain dynamic and thermodynamic parameters (the binding / dissociation equilibrium constant KA / KD, the Gibbs free energy [delta]G degree, the surface coverage [theta], the binding / dissociation rate constant ka / kd and the reaction activation energy Ea) of a nucleic acid aptamer and a ligand thereof on the solid-liquid interface. The method overcomes the defects that a traditional method is expensive in instrument, tedious in operation, single in parameter determination and the like, has the advantages of being high in sensitivity, low in cost, simple and rapid in operation, free of marking and correction, capable of effectively avoiding system errors, capable of determining related parameters at a time and the like, and is suitable for determination of dynamic and thermodynamic parameters of multiple nucleic acid aptamers and ligands thereof.

The invention discloses a method for transforming and screening antibody pH-dependent binding activity. The method comprises the following steps: (1) constructing a wild antibody scFv plasmid template; (2) performing site-specific mutagenesis on six CDR regions of an antibody, and replacing each amino acid in the CDR regions with histidine; (3) establishing a Capture-ELISA condition and carrying out high-throughput screening; and (4) performing KD-ELISA and FACS detection: carrying out gradient dilution on a sample, carrying out KD-ELISA detection by using buffers with different pH values, anddetermining the pH-dependent binding activity of targets cloned on the antigen surface and cell surface according to a curve drawn by detection data. The method provided by the invention can be usedfor obtaining the antibody with pH-dependent binding activity in a high-flux, high-efficiency and low-cost manner.

The present invention relates to a method for calibrating a multiplex assay, comprising: adding a calibration reagent to a solid phase on which a plurality of capturing agents are immobilised, adding a detection molecule which has a capacity to bind to the calibration reagent, detecting bound detection molecule, thereby creating a calibration curve, wherein the calibration reagent comprises at least two different binding molecules, wherein each binding molecule has a capacity to bind specifically to a capturing agent immobilised on the solid phase and a capacity to bind to a detection molecule. Further provided is a multiplex assay system comprising such a calibration reagent.

The invention discloses a method for visually detecting substance concentration based on a mobile exchange interface. The method mainly comprises the steps of 1) enabling different ligands to be subjected to a replacement reaction on the surface of a solid-phase substance (such as nano-particles and cells) to form an optical signal; (2) fixing the solid-phase substance in an electrophoresis channel in advance by using gel (or a self-filling mode and the like), driving a to-be-detected substance to move in the channel in an electrophoresis mode, and replacing a ligand on the surface of the solid-phase substance to form a mobile exchange interface; and 3) recording the interface movement condition, establishing a standard curve of the interface migration rate, the interface width and the substance concentration, and calculating the substance concentration (or the ligand replacement reaction rate) of the reaction on the surface of the solid-phase substance. The method has the advantages of simple operation flow, low cost, visualization and no need of dependence on expensive instruments.

The invention discloses a single-chain antibody (scFv) directly recognizing 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), its preparation method and application. The single-chain antibody includes a heavy chain variable region and a light chain variable region, the sequences of which are shown in SEQ ID NO. The sequences shown have the same functional amino acid sequence. The binding of the single-chain antibody scFv provided by the invention to the antigen can be competitively inhibited by the free hapten, IC 50 The detection limit is 72.93ng / mL, and the detection limit is 13.35ng / mL, which has good antigen binding activity; the linear detection range is between 24.98~212.92ng / mL through the standard curve. The genetically engineered antibody has good application prospects in the immunodetection of AMOZ and the rapid detection and screening of a large number of samples.

The invention discloses a method of detecting an extracellular vesicle surface PD-L1 protein by aptamer of a programmed death receptor-ligand 1 (PD-L1). The method comprises the following steps: 1) asample mixture of a PD-L1 aptamer with a constant concentration and extracellular vesicles with different concentrations is prepared; 2) the sample mixture is placed in a microscale thermophoresis (MST) device for measurement; and 3) the fluorescence of each sample mixture with a different extracellular vesicle concentration is measured, drawing is carried out on the obtained normalized fluorescence in relative to time, an MST trajectory is fit, drawing is then carried out on the normalized fluorescence of the MST trajectory in relative to the extracellular vesicles, a binding curve associatedwith the extracellular vesicle concentration and the normalized fluorescence is fit, and thus, extracellular vesicle surface PD-L1 protein detection is realized. Compared with the prior art, the method disclosed in the invention has the advantages that the sensitivity is high, the operation is simple and convenient, the detection is quick and efficient, few samples are consumed, an economic and affordable price is realized, and the potential for wide promotion is realized.

The invention discloses a quantitative determination method for a specific IgM antibody in plasma, and belongs to the field of antibody detection. The method comprises the following steps: 1) dividing one plasma sample into two groups as to-be-detected plasma and to-be-treated contrast plasma; (2) incubating excessive antigens and to-be-treated control plasma, and neutralizing to-be-detected antibodies in the to-be-treated control plasma to obtain control plasma; 3) taking to-be-detected plasma and the control plasma as samples, combining the to-be-detected plasma and the contrast plasma with corresponding antigens connected to the solid-phase carrier, successively combining the antigen-antibody complex with the IgM antibody and the signal molecule to generate quantifiable data, and subtracting two groups of quantifiable data to obtain detection data; (4) diluting the mixed plasma of thousands of persons as a standard substance into different concentrations according to the steps (1)-(3) to make a standard curve; and 5) substituting detection data of a sample to be detected into the standard curve to obtain the content of the specific IgM antibody in the plasma. According to the determination method, the influence of non-specific binding can be eliminated, and the detection result is reliable.