WO2008128352A1 - Procédés et compositions pour amplifier un signal - Google Patents

Procédés et compositions pour amplifier un signal Download PDF

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Publication number
WO2008128352A1
WO2008128352A1 PCT/CA2008/000750 CA2008000750W WO2008128352A1 WO 2008128352 A1 WO2008128352 A1 WO 2008128352A1 CA 2008000750 W CA2008000750 W CA 2008000750W WO 2008128352 A1 WO2008128352 A1 WO 2008128352A1
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WIPO (PCT)
Prior art keywords
binding agent
analyte
agent
protein
binding
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PCT/CA2008/000750
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English (en)
Inventor
Jean-Francois Houle (Jf)
Irene Chen
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Axela, Inc.
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Publication of WO2008128352A1 publication Critical patent/WO2008128352A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms

Definitions

  • the invention relates to the fields of analyte detection and signal amplification.
  • Detection technologies are often limited by their inability to detect analytes that may be present in the sample at low concentrations.
  • Diffraction-based sensing provides a simple and sensitive method for the detection of biomolecular interactions. Unlike other technologies, grating-based methods do not require the use of fluorescent labels, and the detection beam never passes through the sample.
  • diffraction-based technologies are limited by their ability to detect analytes present at low concentrations in a given sample. There is a need in the existing art for new methods of detecting analytes at low concentrations.
  • the invention features methods, compositions, and kits for detecting analytes at low concentrations.
  • the invention is particularly useful for detecting the presence of an analyte in a biological sample at low (e.g., femtomolar) concentrations.
  • the invention features a method of detecting an analyte in a sample, wherein the method includes contacting a sample with a device having an immobilized binding agent capable of binding to an analyte to form a first complex containing the analyte bound to the immobilized binding agent; contacting the first complex with a first catalytic binding agent that binds to the analyte to form a second complex containing the immobilized binding agent, the analyte, and the first catalytic binding agent; contacting the second complex with a labeling agent, wherein the first catalytic binding agent activates the labeling agent, which then binds to the second complex or the device adjacent to the second complex; introducing into the device a second binding agent that binds to the labeling agent and amplifies a signal associated with the analyte; and detecting the signal.
  • the analyte, first and second binding agents, and labeling agent of the method may be added to the immobilized binding agent on the surface of the device sequentially or as a homogenous mixture.
  • the signal may be detected by measuring, e.g., optical diffraction, absorbance, fluorescence, Raman scattering, phosphorescence, luminescence, radioactivity, or surface plasmon resonance (SPR).
  • the immobilized binding agent of the invention may be protein (e.g., avidin, streptavidin, Protein G, or an antibody) or nucleic acid.
  • the immobilized binding agent may be disposed onto the surface of the device in a pattern capable of optical diffraction.
  • the first catalytic binding agent and second binding agent may include protein (e.g., an antibody or an enzyme) or a nucleic acid.
  • the enzyme may be horseradish peroxidase (HRP), alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-lactamase, esterase, or urease.
  • the labeling agent of the invention may be activated to react with an amino acid side chain (e.g., tyrosine) or a nucleic acid.
  • Exemplary labeling agents include tyramide-dinitrophenyl, biotin tyramine, p-hydroxyphenylpropionyl-biocytin, fluorescein-tyramine, tyramide gold, tyramide polystyrene, tyramide protein devoid of tyrosine, and tyramide nanoparticles.
  • the second binding agent may be a catalytic binding agent.
  • the catalytic second binding agent may be contacted with a precipitating substrate. Contacting the second binding agent with the precipitating substrate amplifies the signal produced upon analyte binding to the surface of the device.
  • Exemplary precipitating substrates include tetramethylbenzidene, 2,2-azino-di(3- ethylbenzthiazoline) sulfonic acid (ABTS), p-nitrophenyl phosphate (pNPP), 5- bromo-4-choro-3'-indolylphosphate (BCIP), diaminobenzidine (DAB), and orthophenylenediamine (OPD).
  • ABTS 2,2-azino-di(3- ethylbenzthiazoline) sulfonic acid
  • pNPP p-nitrophenyl phosphate
  • BCIP 5- bromo-4-choro-3'-indolylphosphate
  • DAB diaminobenzidine
  • OPD orthophenylenediamine
  • the second binding agent and labeling agent may be the same molecule.
  • the labeling agent may be conjugated (e.g., via a covalent bond) to the second binding agent.
  • the analyte of the invention is, e.g., DNA, RNA, proteins, hormones, lipids, virions, or cells.
  • exemplary analytes include C-peptide, glycated hemoglobin, a lipoprotein, low-density lipoprotein (LDL), high-density lipoprotein (HDL), a cytokine, interleukin-6 (IL-6), thyroid- stimulating protein (TSH), anti-thyroid peroxidase (TPO) antibody, a hormone, C-reactive protein (CRP), gelsolin, and copeptin.
  • the sample containing the analyte may be serum, plasma, crude cell lysates, or urine.
  • Figure 1 shows the TMB response when different concentrations of NTproBNP are used in the assay.
  • Rl data was collected from replicate 1 ;
  • R2 data was collected from replicate 2.
  • A A full-scale response curve is shown.
  • B An expanded scale of part (A) is shown, illustrating the distinct rates between different concentrations.
  • Figure 2 shows raw sensor data from all four concentrations of NTproBNP assayed, where (A) is 10 pg/ml, (B) is 1 pg/ml, (C) is 0.1 pg/ml, and (D) is 0 pg/ml.
  • the invention provides methods, compositions, and kits for detecting analytes in a sample, typically present at low concentrations, e.g., femtomolar.
  • the methods of the invention can be used to assess the presence or amounts of various biomarkers in a sample from a subject to facilitate diagnoses of disease and clinical assessments.
  • the methods of the invention include contacting a sample with a device having an immobilized binding agent.
  • the immobilized binding agent binds to an analyte present in the sample, forming a complex on the surface of the device.
  • the complex bound to the device is contacted with a first catalytic binding agent that binds to the analyte.
  • the complex thus formed on the surface of the device is contacted with a labeling agent.
  • the first catalytic binding agent of the complex activates the labeling agent, which binds to the complex or adjacent to the complex.
  • the device is also contacted with a second binding agent that binds to the labeling agent.
  • the second binding agent can amplify a signal associated with an analyte.
  • the signal may be detected using detection methods known to those skilled in the art. e.g., optical diffraction, absorbance, fluorescence, Raman scattering, phosphorescence, luminescence, radioactivity, or surface plasmon resonance.
  • analytes include biomarkers and biomolecules, e.g., hormones, metabolites, DNA, RNA, microRNA, polynucleotides and their analogs, proteins, lipids, toxins, or drugs, as well as larger assemblies, such as virions or cells.
  • biomarkers and biomolecules e.g., hormones, metabolites, DNA, RNA, microRNA, polynucleotides and their analogs, proteins, lipids, toxins, or drugs, as well as larger assemblies, such as virions or cells.
  • Biomarkers including, e.g., C-peptide, glycated hemoglobin, a lipoprotein, low- density lipoprotein (LDL), high-density lipoprotein (HDL), a cytokine, interleukin-6 (IL-6), thyroid- stimulating protein (TSH), anti-thyroid peroxidase (TPO) antibodies, a hormone, C-reactive protein (CRP), gelsolin, and copeptin, may be detected by methods of the invention.
  • the analytes may be present in a biological sample, e.g.. serum, plasma, crude cell lysates, or urine.
  • concentrations of analytes may be detected and measured by the methods described herein.
  • Analytes present at concentrations less than, e.g., 100 milligrams/milliliter, 10 milligrams/milliliter, 1 milligram/milliliter, 100 micrograms/milliliter, 10 micrograms/milliliter. 1 microgram/milliliter, 100 nanograms/milliliter, 10 nanograms/milliliter. 1 nanogram/milliliter, 100 picograms/milliliter, 10 picograms/milliliter, 1 picogram/milliliter, 100 femtograms/milliliter, or 10 femtograms/milliliter, may be detected and measured.
  • the device of the invention contains immobilized binding agents at its surface.
  • the immobilized binding agent may include any substance capable of binding an analyte, such as a protein (e.g., an antibody or fragment thereof, avidin, streptavidin, Protein G, or Protein A) or a polynucleotide.
  • the polynucleotide may possess sequence specificity for the analyte or may be an aptamer.
  • Other binding agents include, e.g., charged polymers, hydrophobic polymers, and carbohydrates.
  • the binding agent immobilized in the device will ultimately depend on the analyte being assayed. Analytes may bind covalently or non-covalently to the immobilized binding agent.
  • the immobilized binding agents may be immobilized in a device by known methods, such as a biotin-avidin or biotin- streptavidin interaction, a Protein G interaction, a GAM-Fc interaction, an amide bond, or through any other covalent or non-covalent interaction.
  • the method may be scaled to detect or measure two. three, four, or more analytes in a sample simultaneously by using devices having two, three, four, or more immobilized binding agents.
  • immobilized binding agents used in the devices of the invention may be agents that are, e.g., magnetic, positively charged, negatively charged, polarized, or capable of forming temporary dipoles, so that the immobilized binding agent can bind analytes in a sample by non-covalent means.
  • the first catalytic binding agent of the invention may be, e.g., a protein or nucleic acid.
  • the first catalytic binding agent is a protein, wherein the protein is an antibody conjugated to an enzyme.
  • the enzyme may be horseradish peroxidase (HRP), alkaline phosphatase, acid phosphatase, glucose oxidase, beta- galactosidase, beta-lactamase, esterase, or urease.
  • the second binding agent may be, e.g., a protein or nucleic acid.
  • the second binding agent may also be catalytic.
  • the second binding agent may be a protein, e.g., an antibody conjugated to an enzyme such as HRP, alkaline phosphatase, acid phosphatase, glucose oxidase, beta- galactosidase, beta- lactamase, esterase, or urease.
  • an enzyme such as HRP, alkaline phosphatase, acid phosphatase, glucose oxidase, beta- galactosidase, beta- lactamase, esterase, or urease.
  • a catalytic binding agent may be intrinsically catalytic or may be catalytic upon conjugation to a catalyst.
  • exemplary catalysts include, e.g., enzymes, ribozymes, and transition metals. Other catalysts are known to those skilled in the art.
  • Amplification of a signal produced upon analyte binding may be achieved by activating a labeling agent that binds to the complex or that binds adjacent to the complex on the surface of the device.
  • the labeling agent may be, e.g., tyramide- dinitrophenyl, biotin tyramine, p-hydroxyphenylpropionyl-biocytin, fluorescein- tyramine, tyramide gold, tyramide polystyrene, tyramide protein devoid of tyrosine, or tyramide nanoparticles.
  • the labeling agent may be activated to react with the complex, or any component thereof, or the surface of the device adjacent to the complex.
  • the labeling agent may, for example, react with tyrosine or another amino acid side chain.
  • the labeling agent may also react with a nucleic acid, e.g., DNA or RNA, the analyte, the immobilized binding agent, or the material to which the immobilized binding agent is bound.
  • amplification of the signal produced upon analyte binding may be achieved by adding a precipitating substrate.
  • the precipitating substrate may be selected from tetramethylbenzidene (TMB), 2,2- azino-di(3-ethylbenzthiazoline) sulfonic acid (ABTS), p-nitrophenyl phosphate (pNPP), 5-bromo-4-choro-3 ' -indolylphosphate (BCIP), diaminobenzidine (DAB), and orthophenylenediamine (OPD).
  • TMB tetramethylbenzidene
  • ABTS 2,2- azino-di(3-ethylbenzthiazoline) sulfonic acid
  • pNPP p-nitrophenyl phosphate
  • BCIP 5-bromo-4-choro-3 ' -indolylphosphate
  • DAB diaminobenzidine
  • OPD orthophenylenediamine
  • the second binding agent may amplify the signal in the absence of a precipitating substrate.
  • Signal amplification by addition of the second binding agent may be the result of, e.g., a gold or polystyrene nanoparticle connected to the second binding agent, fluorophores connected to the second binding agent, or a radioactive-label connected to the second binding agent.
  • the second binding agent is connected to any moiety that may increase optical diffraction.
  • the steps of the method can be repeated several times to provide multiple catalytic binding agents for each analyte bound to the surface of the sensor, thus amplifying the detection signal by one, two, or three orders of magnitude.
  • Other labeling agents and precipitating substrates that would catalyze the deposition of large (e.g., diffraction-enhancing) moieties onto the surface of the device and enhance the binding signal may also be used.
  • the synergistic effect of using a labeling agent and/or a precipitating substrate allows for significant enhancement in the sensitivity of the method used to detect the signal produced upon analyte binding.
  • the signal produced upon analyte binding may be detected or measured using any technique known in the art, including, e.g., optical diffraction, absorbance, fluorescence, Raman scattering, phosphorescence, luminescence, radioactivity, or surface plasmon resonance. Exemplary techniques for detection are listed in, e.g., U.S. Patent No. 6.991,938, hereby incorporated by reference.
  • Diffraction-based assays can involve immobilizing a binding agent, e.g., a protein or nucleic acid, in a distinct pattern on the surface of a device.
  • the binding agents within each spot are not randomly distributed, but are immobilized in a pattern, e.g., a series of parallel lines, that produces a diffraction pattern when illuminated with a light.
  • the binding agents may be immobilized, for example, in eight distinct locations or assay spots on the surface of a device. When a flowing stream of sample is introduced into the device, target molecules bind to the assay spots, resulting in an increased diffraction signal.
  • the intensity of the diffraction signal may be used to generate real-time binding curves.
  • the illumination and detection beams never pass through the sample, which is particularly advantageous for the detection of analytes (e.g., proteins) in complex biological samples.
  • the device used in a diffraction-based assay may be, e.g., a flow-through sensor.
  • the patterns on the surface of the device may be created through, e.g.. microlithography, microcontact printing, inkjet writing, robotic spotting, dip pen nanolithography, nanolithograpahy by atomic force microscopy, or near-field optical scanning lithography.
  • the device may be made of any suitable material, e.g., a synthetic polymer (e.g., polystyrene), glass, metal, silicon, or semiconductor.
  • the device employed may be disposable.
  • the invention enables the direct study of multiple biomolecular interactions in parallel including, e.g., protein-protein interactions, nucleic acid interactions, and nucleic acid-protein interactions.
  • the surface of the sensor may be coated with different immobilized binding agents known in the art. Immobilized avidin groups on the surface of the sensor may be used for high-affinity immobilization of biotinylated binding agents, e.g., biotinylated antibodies or biotinylated polynucleotides. Protein G on the surface of the sensor may bind to the Fc region of immunoglobulin molecules, allowing oriented immobilization of antibodies as binding agents on the surface of the device. Goat Anti-Mouse-Fc (GAM-Fc)-coated surfaces bind to the Fc region of mouse antibodies, allowing oriented immobilization of binding agents, e.g., mouse antibodies, on the surface of the device employed by the invention.
  • Immobilized avidin groups on the surface of the sensor may be used for high-affinity immobilization of biotinylated binding agents, e.g., biotinylated antibodies or biotinylated polynucleotides.
  • Immobilized carboxylate groups on an amine-reactive surface may be used to covalently link binding agents, e.g., with amide bonds, to the device's surface via an amine coupling reaction.
  • Proteins, peptides, nucleic acids, and other biomolecules can be immobilized.
  • Other exemplary reactive linking groups e.g., hydrazines, hydroxylamines, thiols, carboxylic acids, epoxides, trialkoxysilanes, dialkoxysilanes, and chlorosilanes may be attached to the surface of the device, such that binding agents may form chemical bonds with those linking groups to immobilize them on the surface of the device.
  • a variety of detection methods may be used with the methods of the invention to detect or measure the concentration of an analyte.
  • Surface-enhanced Raman spectroscopy (SERS) or infrared (IR) spectroscopy may be used.
  • Other optical techniques that may be applied to the methods of the invention are fluorescence or phosphorescence.
  • Non-optical techniques e.g., voltametric or amperometric methods, may also be used.
  • SPRS Surface plasmon resonance spectroscopy
  • FTR frustrated total reflection
  • evanescent wave ellipsometry scattered total internal reflection
  • optical wave guide sensors evanescent wave based imaging, e.g., critical angle resolved imaging, luminescence, and absorbance are other methods that may be used for the detection of analytes in the methods of the invention described herein.
  • the methods of the invention may be used for the quantitative measurement of analytes across a broad dynamic range, from femtomolar to millimolar concentrations.
  • the methods of the invention permit a wide range of applications including, e.g., aggregation studies, substrate-activity measurements, enzyme inhibition studies, monitoring levels of biomarkers (with and without disease relevance), and the detection of large species such as viral particles, microorganisms, and cells.
  • the methods described herein may be used to diagnose a disease (e.g., cancer, an autoimmune disease, diabetes, or an infection) in a subject.
  • a disease e.g., cancer, an autoimmune disease, diabetes, or an infection
  • the methods of the invention may be used to diagnose a disease in a subject that results in the expression or production of a particular analyte.
  • a diagnosis may be made if, for example, the presence of the analyte is detected in a biological sample obtained from the subject.
  • the disease being diagnosed may be cancer (e.g., a carcinoma, lymphoma, blastoma, sarcoma, or leukemia). More particular examples of such cancers include, e.g., prostate cancer, squamous cell cancer, small-cell lung cancer, non-small-cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, vulval cancer, thyroid cancer, hepatic carcinoma, gastric cancer, melanoma, and various types of head and neck cancer.
  • cancers e.g., a carcinoma, lymphoma, blastoma, sarcoma, or leukemia.
  • the disease may be, e.g., an autoimmune disease.
  • the autoimmune disease being diagnosed may be, e.g., autoimmune hepatitis, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, type I diabetes, rheumatoid arthritis, psoriasis, Hashimoto's thyroiditis, Graves' disease, Sjogren's syndrome, or scleroderma.
  • infections e.g., bacterial or viral infections
  • exemplary bacteria and viruses that may lead to an infection include hepatitis C, human immunodeficiency virus (HIV), adenovirus type 2 hexon, Aspergillus fumigatus, Borrelia afzelii, Borrelia garninii, Campylobacter jejuni, Candida albicans, Chlamydia, coxsackievirus B l , coxsackievirus B5. coxsackievirus B6, cytomegalovirus, Echinococcus.
  • HIV human immunodeficiency virus
  • adenovirus type 2 hexon Aspergillus fumigatus
  • Borrelia afzelii Borrelia garninii
  • Campylobacter jejuni Candida albicans
  • Chlamydia coxsackievirus B l
  • Physicians and researchers may use the methods of the invention described herein to detect plasma biomarkers, e.g., lipoproteins or cardiac biomarkers or monitor thyroid function. Additionally, physicians and researchers may use the methods of the invention to detect hormones to assess various physical conditions, such as pregnancy, ovulation, menopause, and diseases such as cancer. Physicians and researchers may also use the invention to detect various cytokines, which are initially detected at picomolar levels, and other biomarkers (e.g., CRP).
  • CRP biomarkers
  • the methods of the invention speed the detection of an analyte in a number of ways including, e.g., quantifying analyte concentration and purity, characterizing binding kinetics, determining specificity and cross-reactivity, optimizing analyte concentrations, step times, buffers, and additive composition, monitoring assay performance and matrix effects, and multiplexing analytes with minimized interference.
  • a dotLabTM avidin-functionalized optical diffraction device (Axela Inc., Canada) was used to detect a cardiac biomarker, the N-terminal portion of proBNP (brain natriuretic peptide) (NTproBNP).
  • the exemplary experiments involved pre- mixing all assay reagents to prepare a complex or "sandwich" (Table 1). The antibody concentrations outlined in Table 1 were mixed with 0 pg/ml, 0.1 pg/ml, 1 pg/ml, and 10 pg/ml of NTproBNP in vials and incubated overnight at 4°C. Different avidin-functionalized optical diffraction devices were then contacted with the sandwiches. The dinitophenyl (DNP) amplification reagent was subsequently added to the sensor, followed by anti-DNP-HRP antibody. Finally, TMB was added to the sensor. Detailed assay conditions are listed in Table 2. Table 1
  • the tyramide signal amplification (TSA) kit was purchased from Perkin Elmer (Cat. No. NEL747B) and included a concentrated DNP amplification reagent, DNP amplification diluents, and anti-DNP-HRP antibody that was diluted in BSA- Tween-20 (BSA-T).
  • BSA-T BSA- Tween-20
  • the concentrated DNP amplification reagent was diluted in 150 ⁇ l of dimethyl sulfoxide (DMSO) and stored as a stock solution. The working solution was diluted 1 :50 in the DNP amplification diluents just prior to the assay and discarded after use.
  • DMSO dimethyl sulfoxide
  • the anti-DNP- HRP antibody was diluted 1 : 100 in 5 mg/ml BSA in PBS supplemented with 0.025% Tween-20 (PBS-T) and stored at 4°C until use.
  • TrueBlue TMB was used as the precipitating substrate.
  • the buffer system was PBS supplemented with 0.025% Tween-20 and the blocking reagent was 5 mg/ml BSA in PBS supplemented with 0.025% Tween-20.
  • the TMB signal reached the limit of the detector within 100 seconds, and at 1 pg/ml of NTproBNP, the TMB signal reached the limit of the detector in 700 seconds.
  • a 5 pg/ml sample of NTproBNP in the sandwich resulted in 0.04 diffraction intensity (DI) signal in the first 100 seconds.
  • DI diffraction intensity
  • FIGs 1 and 2 illustrate the enhanced sensitivity of detection using both TSA and TMB. Note that the TMB signal in Figure 1 was normalized to aid comparison. Figure 2 shows representative traces of the experiments performed to demonstrate the added sensitivity of using TSA combined with precipitating TMB.

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Abstract

La présente invention concerne des procédés, des compositions, et des trousses pour détecter un analyte à de basses concentrations dans un échantillon par diffraction optique et un procédé d'amplification de signal en deux étapes. Les procédés de l'invention peuvent être utilisés pour déterminer la présence de divers biomarqueurs dans un échantillon provenant d'un sujet et faciliter le diagnostic d'une maladie et ses évaluations cliniques.
PCT/CA2008/000750 2007-04-19 2008-04-21 Procédés et compositions pour amplifier un signal WO2008128352A1 (fr)

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CN103336115A (zh) * 2013-07-17 2013-10-02 江阴泽成生物技术有限公司 一种人甲状腺过氧化物酶抗体（TPOAb）的磁微粒化学发光免疫分析试剂盒及其检测方法
WO2013148498A1 (fr) * 2012-03-27 2013-10-03 Ventana Medical Systems, Inc. Conjugués de signalisation, et procédés d'utilisation
JP2015135338A (ja) * 2008-10-31 2015-07-27 ベー．エル．アー．ハー．エム．エス ゲゼルシャフト ミット ベシュレンクテル ハフツング 糖尿病の予測バイオマーカーとしてのアルギニンバソプレシンプロホルモン
WO2016061460A1 (fr) * 2014-10-17 2016-04-21 Carnegie Mellon University Dosages améliorés pour la détection de biomolécules basés sur l'amplification du signal par tyramide et des sondes de gamma-pna
WO2018148461A1 (fr) * 2017-02-09 2018-08-16 Essenlix Corp. Dosage avec amplification
US10778913B2 (en) 2013-03-12 2020-09-15 Ventana Medical Systems, Inc. Digitally enhanced microscopy for multiplexed histology
CN113195731A (zh) * 2018-12-17 2021-07-30 豪夫迈·罗氏有限公司 灵敏的葡萄糖测定法
CN114235781A (zh) * 2021-12-22 2022-03-25 上海海洋大学 基于表面增强拉曼光谱技术定量检测海水中β-半乳糖苷酶的方法
US11567008B2 (en) 2018-10-31 2023-01-31 Sony Corporation Immunostaining method, immunostaining system, and immunostaining kit
US11712177B2 (en) 2019-08-12 2023-08-01 Essenlix Corporation Assay with textured surface

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