46 results about "Dissociation constant" patented technology

The invention provides a nucleic acid aptamer for specific recognition of morphine and application of the nucleic acid aptamer, and belongs to the technical field of biosensing and detection. The nucleotide sequence of the nucleic acid aptamer is as shown in SEQ ID NO: 1. The screening method is based on a Capture-SELEX technology and comprises the key steps that streptavidin magnetic beads are used for fixing an ssDNA library, estradiol, deabietic acid and totarol are introduced to serve as reverse screening substances so as to remove non-specific sequences, and finally the high-specificity aptamer is obtained through high-throughput sequencing and affinity determination. The dissociation constant of the aptamer and morphine is 127.31 nM, and the aptamer shows high affinity and high specificity. The invention further relates to application of the aptamer in preparation of a sensor and a kit for detecting morphine, and a new technical means is provided for rapid detection of morphine.

The invention discloses CML and CEL aptamers screened based on an efficient microfluidic SELEX platform, and belongs to the technical field of food safety biology. Through the microfluidic SELEX platform integrated with library fixation, target and library incubation, magnetic separation, fluorescence monitoring and other functions, the capsule SELEX screening method with less magnetic bead consumption, higher library immobilization rate, shorter screening time and higher enrichment efficiency is realized; the requirements and limitation of methods such as qPCR (quantitative polymerase chain reaction) and fluorescence ratio determination on specific instruments in the prior art are broken through; finally, the aptamer S2 with excellent performance of CML and CEL is obtained for the first time through the screening method, truncation optimization is carried out based on the combination mechanism of the aptamer S2, the aptamer S2-40 with shorter sequence and more excellent performance is obtained, and the dissociation constants measured by an ITC method and an SGI method are 6.65 + / -3.07 mu M and 42.1 + / -19.34 nM.

HPV16 is a main cause of malignant tumors such as cervical cancer, and E6 protein of HPV16 becomes a key treatment and diagnosis target. In the prior art, an HPV16 detection antibody has the problems of poor stability, dependence on cold chain transportation, low preparation efficiency and the like. According to the invention, a nano antibody sequence which is high in affinity (dissociation constant KD is less than or equal to 1 * 10 <-7 > M) and stable at room temperature is obtained through total synthesis library construction and a phage-assisted directed evolution (PACE) screening technology. After the antibody is stored at 25 DEG C for 30 days, the activity of the antibody is kept to be greater than or equal to 95%, the antibody can be directly used for household test paper, the accuracy rate reaches 98%, and meanwhile, the antibody can be expanded to medical applications such as CAR-T cell therapy. Compared with a traditional antibody, the antibody has the advantages of being short in preparation period, low in cost, wide in application scene and the like, and a breakthrough solution is provided for early screening of cervical cancer.

The invention discloses a high-stability PRSSV-GP5 nano antibody as well as a preparation method and application thereof, and belongs to the technical field of PRSSV vaccines. The PRSSV-GP5 nano antibody provided by the invention has strong affinity with the antigen PRSSV-GP5 and high stability, the equilibrium dissociation constant Kd is 3.84 * 10 <-7 > M, the stoichiometric N is 1.13, the enthalpy change delta H is-389.24 KJ / mol, and the entropy change delta S is-1183.35 J / mol.K; the optimal concentration of the polypeptide and the antigen is 5 mu g / mL, and the polypeptide still has certain binding capacity with GP5 protein when the concentration is 0.005 mu g / mL; the preparation method of the PRSSV-GP5 nano antibody provided by the invention is simple and easy to operate, and can be used for preparing the PRSSV-GP5 nano antibody which is strong in antigen affinity, high in stability and low in binding concentration.

The invention relates to an aptamer specifically bound with aflatoxin B1 and application of the aptamer, and belongs to the technical field of biological detection. According to the invention, aflatoxin B1 is taken as a target, on the basis of broad-spectrum recognition of aflatoxin (aflatoxin B1, aflatoxin B2, aflatoxin M1 and aflatoxin G1) by an original aptamer, an aptamer sequence is modified by using directed evolution, and the aflatoxin B1, aflatoxin B2, aflatoxin M1 and aflatoxin G1 are identified on the basis of molecular dynamics in combination with free energy. And the aptamer sequence for recognizing the aflatoxin B1 with high affinity and specificity is obtained. The dissociation constant of the aptamer to the aflatoxin B1 is 9.2 nM, and the aptamer has high affinity to the aflatoxin B1; in addition, the aptamer provided by the invention can only detect the aflatoxin B1, has no recognition force on the aflatoxin B2, the aflatoxin M1, the aflatoxin G1, the fumonisins B1, the zearalenone and the ochratoxin A, and has good specificity.

The invention discloses a screening method and application of a shark-derived anti-idiotypic nano antibody, and belongs to the technical field of biological pharmacy and vaccines. The screening method comprises the following steps: immunizing adult shark by using a target pathogen specific antigen, collecting peripheral blood, separating lymphocytes, extracting RNA (Ribonucleic Acid), carrying out reverse transcription to synthesize cDNA (Complementary Deoxyribonucleic Acid), amplifying a nano antibody variable region fragment of a shark IgNAR (Immunoglobulin Nucleic Acid Receptor) gene, and constructing a phage display gene bank of which the storage capacity is greater than or equal to 1 * 10 < 8 > CFU and the recombination rate is greater than or equal to 85%; a variable region of a target pathogen specific antibody is taken as a target, specific bacteriophage is enriched through 3-4 rounds of gradient washing and elutriation (Tween-20 concentration and washing time are gradually improved), and the shark source anti-idiotypic nano antibody with high affinity and high purity (greater than or equal to 95%) is obtained by combining ELISA screening positive clone, gene sequencing de-weighting and SPR detection (combining dissociation constant KDlt, 1 * 10 <-7 > mol / L).

The present invention discloses a highly stable PRSSV-GP5 nanobody and a preparation method and application thereof, and belongs to the technical field of PRSSV vaccine. The PRSSV-GP5 nanobody provided by the present invention has a strong affinity and high stability for the antigen PRSSV-GP5, and the equilibrium dissociation constant Kd is 3.84×10 ‑7 M, the stoichiometric N is 1.13, the enthalpy change ΔH is ‑389.24KJ / mol, and the entropy change ΔS is ‑1183.35J / mol·K; the optimal concentration with the antigen is 5μg / mL, and it still has a certain binding ability with the GP5 protein at a concentration of 0.005μg / mL; the preparation method of the PRSSV‑GP5 nanoantibody provided is simple and easy to operate, and can be used for the preparation of PRSSV‑GP5 nanoantibodies with strong antigen affinity, high stability and low binding concentration.

An aptamer of nattokinase and method for screening the aptamer are provided, which relate to technical fields of biotechnology. A set of nattokinase nucleic acid aptamers screened by capillary electrophoresis separation technology is: SEQ ID NO:1˜SEQ ID NO:17. The dissociation constants of the seven aptamers were detected by surface plasmon resonance technology, and the affinity of the seven aptamers was strong, with affinities between 8.7-87 nM. The nucleic acid aptamer of the present disclosure has precise specificity, high affinity, and is convenient for chemical modification, and can be used as an effective molecular recognition tool for the high-sensitive analysis of proteins. it. The recognition technology based on nucleic acid aptamers provides a basis for the development of NK determination and efficient separation and purification.

The present application relates to an assay method using affinity crosslinking of DNA-linked ligands to proteins to enable small molecule screening and determination of apparent affinity constants of ligands to the proteins. This method has been applied to determine 96 compounds' dissociation constants to a protein target simultaneously, and directly determine a compound's IC50 against five protein targets concurrently in crude cell lysates. Additionally, this approach was used to screen a Library of Pharmacologically Active Compounds (LOPAC) library against dihydrofolate reductase (eDHFR), enabling the discovery of a novel eDHFR inhibitor (IC50=7.9 μM). An assay kit and the method of uses are within the scope of this disclosure.

The application discloses a nucleic acid aptamer of maytansine and application thereof, the nucleic acid aptamer is Seq2-X, and a DNA sequence of Seq2-X is shown as SEQ ID NO. 1. The nucleic acid aptamer can be combined with the target maytansine with high affinity and high specificity, the equilibrium dissociation constant is in the nanomolar range, the water solubility is good, and after being combined with the fat-soluble maytansine, the solubility of the drug can be improved. By utilizing the specific interaction between the nucleic acid aptamer and maytansine, the stability of maytansine drug can be improved, the cytotoxicity of maytansine is enhanced, and the treatment window is widened. By utilizing the nucleic acid aptamer as a drug delivery carrier of maytansine, the limitation of maytansine in clinical application can be improved, which has important significance for the design and development of maytansine drugs in clinical application.

The embodiment of the invention discloses a method for screening a tumor immune checkpoint inhibitor based on electrochemical luminescence sensing. According to the invention, an electrochemiluminescence sensor is established, and resonance energy transfer between luminol (an electrochemiluminescence donor) and a PD-L1-targeting metal cluster (an electrochemiluminescence receptor) is utilized; the electrochemical luminescence sensor capable of rapidly, sensitively, simply, conveniently and quantitatively analyzing the equilibrium dissociation constant between the non-antibody immune checkpoint inhibitor and the PD-L1 expressed on the tumor cell membrane at low cost is established. The efficiency of blocking the PD-1 / PD-L1 by the inhibitor is evaluated through quantitative analysis of the constant, so that the potential non-antibody immune checkpoint inhibitor is effectively screened.

The present invention related to methods for determining and correcting the dissociation constant KD of a binding pair of two molecules. The method measures of the binding of two biomolecules at 4 or more different absolute temperatures (T) between 4- 40ºC and then calculating and correcting the dissociation constants (KD).

The invention discloses a nucleic acid aptamer targeting aflatoxin B1 as well as an acquisition method and application of the nucleic acid aptamer. The relative molecular weight of the aflatoxin B1 is only 312.27, and the molecular structure is lack of active groups for directional coupling under mild conditions, so that the aflatoxin B1 is difficult to fix on the surface of a solid-phase carrier on the premise of not damaging the main conformation or key recognition sites of the aflatoxin B1. According to the invention, an optimized Capture-SELEX screening method is adopted, so that not only is the nucleic acid aptamer targeting aflatoxin B1 successfully screened, but also the dissociation constant (Kd value) of the nucleic acid aptamer measured by an isothermal titration calorimetry is extremely low, which indicates that the target affinity of the nucleic acid aptamer is extremely strong.

The invention discloses a method for screening a beta-ZOL aptamer based on a Capture-SELEX (systematic evolution of ligands by exponential enrichment) technology, which comprises the following steps: hybridizing a biotin labeled capture chain with a random library by Capture-SELEX, and fixing the library on a streptavidin modified solid phase carrier; the library is eluted using a free target, and the weaker or unbound sequences are still retained on the solid phase carrier. The amount of substance of the single-stranded DNA screened in each round is monitored through real-time fluorescent quantitative PCR, all eluent is subjected to PCR amplification, and finally the single-stranded DNA is prepared through a biotin-streptavidin magnetic bead method. And repeating the steps for 11 rounds, finally performing high-throughput sequencing on the enriched library, and determining dissociation constants and specificity by using an affinity evaluation technology to obtain the nucleic acid aptamer of beta-ZOL.

The invention discloses a sequence-based protein-ligand binding and affinity prediction method, which comprises the following steps of: 1, acquiring a prediction data set, and carrying out data processing on the prediction data set; the prediction data set comprises an amino acid sequence of a protein, an SMILE sequence of a ligand, whether the protein and the ligand can be combined with a tag or not, and a protein and ligand binding affinity dissociation constant; step 2, constructing a protein-ligand binding and affinity prediction model; step 3, training the protein-ligand binding and affinity prediction model by using the data set after data processing; and step 4, inputting the amino acid sequence of the protein and the SMILE sequence of the corresponding ligand into the trained protein-ligand binding and affinity prediction model to obtain a prediction result of whether the protein and the ligand can be bound or not and the binding affinity. According to the method, the accuracy and generalization ability of the prediction model and the interpretability of the model are improved.

The application discloses a digoxin nanobody and a preparation method and application thereof. The amino acid and nucleotide sequences of the digoxin nanobody are shown in SEQ ID NO. 8-9. The antibody has good affinity to a target antigen, and the dissociation constant Kd value is about 73.1 nM, and can be used for digoxin content determination, nucleic acid probe detection and in-vivo digoxin neutralization and detoxification. The antibody has the advantages of small volume, high stability, low cost, low immunogenicity and the like, and has a good application prospect.

Bioequivalence and / or bioavailability data are mandatory for filing of NDA and ANDA. Presently in absence of proper procedures estimates of these data through peak plasma level concentrations and time to reach peak plasma levels are used. The method suffers from the serious drawback that peak plasma levels do not correlate with efficacy due to delay in transfer to tissue sites as well as differences in metabolic patterns in drugs. Here we have devised a novel method for determination of bioavailability and bioequivalence from measurement of threshold times and concentrations, intrinsic activity, and dissociation constants in vivo in humans. The method essentially determines Intrinsic activities and threshold doses through measurement of drug responses in humans and then uses a mathematical expression to determine KD values of drugs, in vivo, in humans. The process may be applied to animals as well. The method would go a long way in evaluation of true bioavailability and bioequivalence and would also be useful in design.

This method for detecting a test substance in a sample by a single molecule detection method includes: a complex formation step for forming a complex that includes the test substance, a marker comprising a marker substance and a first probe molecule capable of binding to the test substance, and a capturing body comprising an insoluble carrier and a second probe molecule capable of binding to the test substance; a separation step for separating the marker from the complex; and a detection step for detecting the marker separated from the complex by the single molecule detection method. The equilibrium dissociation constant (KD1) between the test substance and the first probe molecule and the equilibrium dissociation constant (KD2) between the test substance and the second probe molecule are both less than 1.0×10-8 M. The separation step includes a step for heating the complex at 37°C or higher in a processing liquid containing at least one agent selected from the group consisting of a surfactant, a pH modifier, and a reducing agent.

The invention discloses a VHH chain of an anti-TrxA nano antibody and application of the VHH chain, and belongs to the technical field of nano antibodies. The VHH chain of the anti-TrxA nano antibody disclosed by the invention comprises amino acid sequences of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4; according to the anti-TrxA nano antibody obtained by the invention, the dissociation constant (KD) of the nano antibody and a TrxA antigen reaches 2.29 * 10 <-9 > M, and the nano antibody shows excellent binding dynamic characteristics and high affinity. The kit can be used for ELISA and WB detection of TrxA recombinant protein.

An albumin binding constructs that enhances the pharmacokinetic performance of therapeutic peptides and proteins, extending their half-lives and reducing administration frequency, is disclosed. The core peptide DICLPRWGCLW (SEQ ID NO: 1) is covalently linked to hydrophilic, flexible glycine-serine (Gly-Ser) linkers of the formula (ggs)xgg where X is 3 to 8 units. These linkers provide strong albumin binding (dissociation constant <20 nanomolar). Some constructs feature the GGSGGSGGSGGRLIEDICLPRGCLWEDD (SEQ ID NO: 4) peptide and allow fusion of active proteins like Klotho, maintaining biological activity and leveraging albumin's extended half-life. Constructs are produced using cellular expression systems (CHO, HEK293, Transgenic Insect Cells) for scalability. These constructs show prolonged half-lives comparable to at least 90% of native albumin. Klotho constructs using the albumin binder demonstrate FGF23 binding dissociation coefficients between 15-30 nanomolar essentially the same as natural Klotho Designed to minimize antigenicity, these constructs improve drug delivery efficacy and patient outcomes.

The purpose of the present invention is to provide a method for efficiently producing an oligonucleotide by a solid-phase synthesis approach. The present invention provides a method for producing an oligonucleotide by a solid phase synthesis approach, the method comprising a step for reacting an oligonucleotide in which a hydroxyl group at a chain extendable end is protected with a protective group that can be removed under acidic conditions and a deblocking solution to remove a protective group for a hydroxyl group at the chain extendable end, wherein the deblocking solution is a deblocking solution containing a base, an acid, and an aprotic solvent, and the base is a base having an acid dissociation constant (pKa) of a conjugate acid of 5-12, and an oligonucleotide in which the content ratio of the 2', 3'transfer body is a certain amount or less.

The invention relates to the technical field of biological medicines, and provides an aptamer specifically bound with lipophilic polyether toxin as well as a screening method and application of the aptamer. An RNA-Capture SELEX technology optimization scheme is adopted to obtain an RNA aptamer R1852, the RNA aptamer R1852 can be simultaneously combined with OA, DTX-1 and AZA-1 toxins with high affinity, and equilibrium dissociation constants (Kd) are as follows: OA: 2.139 * 10 <-6 > M; dTX-1: 3.755 * 10 <-7 > M; and AZA-1: 2.429 * 10 <-6 > M. The invention also discloses the aptamer. As a molecular recognition probe capable of being specifically combined with OA, DTX-1 and AZA-1 at the same time, the aptamer has the advantages of being high in affinity, high in specificity, good in stability, low in immunogenicity, easy to synthesize, modify and mark and the like. The method can be used for separation, enrichment and detection of trace OA, DTX-1 and AZA-1 in water or aquatic product samples, and rapid detection and removal of OA, DTX-1 and AZA-1 toxins in water or aquatic products are realized.

To provide an improved analysis method or the like capable of analysis using a specific bonding function of DNA.SOLUTION: The analysis method is an analysis method for analyzing a target substance in a sample using the sample that may contain the target substance, the analysis method including: Acquiring a first signal of the sample after the reaction, reacting the sample with a second test substance containing a DNA assembly consisting of DNA of one or more types of sequences and acquiring a second signal of the sample after the reaction, and analyzing the target substance in the sample based on a difference between the first signal and the second signal, wherein a dissociation constant between the DNA assembly and the target substance is larger than a dissociation constant between the aptamer and the target substance.SELECTED DRAWING: Figure 6

The application provides a structure-based evaluation model for equilibrium dissociation constant of a peptide ligand molecule and a target protein, comprising: obtaining of peptide ligand equilibrium dissociation constant data; obtaining of peptide ligand and receptor protein interaction relationship characteristic data; data set for algorithm system construction: construction of a targeting IgG series polypeptide characteristic library and a targeting alpha beta 42 series polypeptide characteristic library, and construction of an independent verification data set; using the targeting IgG series polypeptide characteristic library to construct a machine learning algorithm classifier system, optimizing related parameters, testing by using the targeting alpha beta 42 series polypeptide characteristic library, and evaluating system performance; and evaluating actual prediction performance of the system by using the constructed related independent data set. The application uses a virtual screening method to study a peptide ligand and a target protein interaction region, which can further improve drug screening efficiency and reduce corresponding cost.

The present invention discloses a method for generating stable tautomers and protonation states of drug molecules. This method traverses all possible isomeric states of the molecule through an isomer conversion template, scores each isomeric state using a machine learning potential energy function, and determines the structure of the low-energy tautomer of the molecule. Then, based on the small molecule dissociation constant prediction method, the dissociation constants of each potential dissociation site in each low-energy tautomer are predicted to generate all possible protonation states of each isomer. The present invention integrates four aspects: tautomer conversion, scoring, ranking, and protonation of drug molecules, and can efficiently process small molecule compounds.

The invention belongs to the field of theanine detection, and particularly discloses a theanine nucleic acid aptamer and application of the theanine nucleic acid aptamer in immunological detection. According to the method, an exponential enrichment ligand systematic evolution technology (SELEX) is utilized, carboxyl magnetic beads serve as a target fixing medium, theanine serves as a target, a nucleotide sequence specifically bound with the target is obtained through 14 rounds of screening, the dissociation constant reaches mu M after truncation optimization, and due to the advantages of being high in affinity, capable of being artificially synthesized, low in cost, easy to chemically modify and the like, the target can be specifically bound to the carboxyl magnetic beads. And the antibody can replace the theanine antibody to be used for immunological detection. The invention provides application of a theanine nucleic acid aptamer in immunohistochemical and ELISA (enzyme-linked immunosorbent assay) detection of theanine.

This specification provides antigen-binding protein constructs (ABPCs) that bind to TROP2 and their use. This specification provides antigen-binding protein constructs (ABPCs) and pharmaceutical compositions comprising an effective amount of ABPC, comprising a first antigen-binding domain (ABD) that can specifically bind to TROP2 or an epitope of TROP2 presented on the surface of a target mammalian cell, wherein (a) the dissociation rate of the first ABD at pH about 4.0 to about 6.5 is faster than the dissociation rate at pH about 7.0 to about 8.0, and / or (b) the dissociation constant (KD) of the first ABD at pH about 4.0 to about 6.5 is greater than the KD at pH about 7.0 to about 8.0.

This application relates to the field of tumor immunodiagnostic technology, and particularly to a monoclonal antibody, polynucleotide, preparation method, and application of antiphosphatidylinositol proteoglycan-3; the monoclonal antibody includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes heavy chain region CDR1 with amino acid sequence as shown in SEQ ID NO:1, heavy chain region CDR2 with amino acid sequence as shown in SEQ ID NO:2, and heavy chain region CDR3 with amino acid sequence as shown in SEQ ID NO:3; the light chain variable region includes light chain region CDR1 with amino acid sequence as shown in SEQ ID NO:4, light chain region CDR2 with amino acid sequence as shown in SEQ ID NO:5, and light chain region CDR3 with amino acid sequence as shown in SEQ ID NO:6; the dissociation constant KD(M) of the monoclonal antibody against GPC3 antigen is 1.21E-11, which is a high-affinity N-terminal antibody, suitable for the detection of GPC3 in clinical serum samples.

The present invention belongs to the technical field of antibiotic detection, and specifically relates to an avermectin B1a aptamer and a screening method and application thereof. The nucleic acid sequence of the avermectin B1a aptamer includes a sequence as shown in SEQ ID NO.1. The avermectin B1a aptamer has a dissociation constant of 63.13±8.91nM, has a strong affinity for avermectin B1a, and can specifically recognize avermectin B1a.

The present invention relates to novel albumin binding constructs that enhance the pharmacokinetic performance of therapeutic peptides and proteins, extend their half-life and reduce the frequency of administration. The core peptide DICLPRWGCLW (SEQ ID NO: 1) is covalently linked to a hydrophilic and flexible glycine-serine (Gly-Ser) linker of the formula (ggs) xg, wherein x is from 3 to 8 units. The linkers provide a strong binding force (dissociation constant lt; 20 nanomoles) to albumin. Some constructs have a GGSGGSGGSGGRLIEDICLPRGCLWEDD (SEQ ID NO: 4) peptide, allowing fusion of active proteins, such as Klotho, maintaining biological activity, and utilizing the long half-life of albumin. In some embodiments, constructs are produced using cellular expression systems (CHO cells, HEK293 cells, transgenic insect cells) to ensure scalability. Upon administration, these constructs exhibit an extended half-life up to at least 90% of the half-life of native albumin. The novel Klotho constructs using the novel albumin conjugates show a FGF23 binding dissociation constant of from 15 to 30 nanomoles, substantially the same as that of natural Klotho. These constructs are designed to minimize antigenicity, thereby enhancing drug delivery effectiveness and improving patient prognosis.