WO2003036297A1 - Dosages non immunologiques pour la detection et la determination de la proteine c reactive - Google Patents

Dosages non immunologiques pour la detection et la determination de la proteine c reactive Download PDF

Info

Publication number
WO2003036297A1
WO2003036297A1 PCT/IE2001/000124 IE0100124W WO03036297A1 WO 2003036297 A1 WO2003036297 A1 WO 2003036297A1 IE 0100124 W IE0100124 W IE 0100124W WO 03036297 A1 WO03036297 A1 WO 03036297A1
Authority
WO
WIPO (PCT)
Prior art keywords
crp
label
group
bsa
alkyl
Prior art date
Application number
PCT/IE2001/000124
Other languages
English (en)
Inventor
John Martin Doyle
Kieran Gerard Walshe
Original Assignee
Tridelta Development Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tridelta Development Limited filed Critical Tridelta Development Limited
Priority to PCT/IE2001/000124 priority Critical patent/WO2003036297A1/fr
Publication of WO2003036297A1 publication Critical patent/WO2003036297A1/fr

Links

Classifications

    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4737—C-reactive protein

Definitions

  • This invention relates to a method of detecting or determining C- reactive protein (CRP) in diverse animal species.
  • CRP C-reactive protein
  • Interleukin-1, interleukin-6 (IL-6) and transforming growth factor- ⁇ 6 stimulate the transcriptional activation of CRP and other acute phase proteins.
  • IL-6 which is produced by activated leucocytes, fibroblasts and endothelial cells is the major stimulant for CRP production.
  • CRP is a member of the pentraxin family of proteins and is well conserved biologically throughout evolution. It shows approximately 70% sequence homology with another member of this family Serum Amyloid P (SAP) which is also present in the plasma. No CRP deficiency diseases have ever been identified which suggests that it has a crucial in vivo role.
  • SAP Serum Amyloid P
  • CRP has been detected in all mammals, birds, amphibians and in marine teleosts and is composed of five identical subunits bound non-covalently to each other in a cyclic pentameric structure. Each subunit has 206 amino acids and an approximate molecular weight of 21,000.
  • CRP requires calcium for ligand binding and five calcium dependent ligand binding sites are present on one face of the CRP molecule through which it recognises phosphorylcholine (PC, a hydrophilic portion of phosphatidylcholine on pneumococcal C polysaccharide) and other bacterial products. Also present on the CRP molecule is a hydrophobic pocket which accommodates the methyl groups of phosphorylcholine. PC is also found in phospholipids in cell membranes and plasma lipoproteins and in the complex polysaccharides of plants, fungi and bacteria.
  • CRP has two major biological functions: (i) A role in the innate immune response against infection: Here, CRP activates complement by the classical C ⁇ q pathway and reacts with F c ⁇ receptor and possibly other receptors on phagocytic cells. CRP activation of the classical C ⁇ q pathway differs from IgG activation of the pathway in that it is localised to the collagen-like regions instead of the globular head groups of C ⁇ q . CRP is most efficient at early complement pathway activation, (ii) Removal of membrane and nuclear material from necrotic cells: CRP binds to damaged tissue, to nuclear antigens and to certain pathogenic organisms in a calcium dependent manner.
  • the calcium sites react with components including phosphatidylcholine and sphingomyelin on damaged cell membranes when exposed during injury. CRP also binds to chromatin which has been exposed during necrosis or apoptosis and to small ribonuclear proteins on damaged cell membranes.
  • CRP detection has been used for many years in the diagnosis of tissue injury or inflammation as high concentrations can be found in patients suffering from infection, inflammation, trauma, malignancies, stress, arthritis, surgery and acute myocardial infarction. CRP detection is also found to be useful in monitoring efficacy of therapy to the aforementioned diseases.
  • CRP cardiovascular risk factor
  • Troponin T and I were used as markers for patients with acute coronary syndrome.
  • Troponin T is still found to be a more accurate predictor than CRP in the first 72 hours of illness however CRP can be used independently to predict both cardiac risk and coronary revascularisation during a six month follow up period. It has been shown in studies which had in- hospital death and acute myocardial infarction as end-points, it was found that CRP was not predictive (Heeschen, C. et al., (2000) Journal of the American College of Cardiology 35 (5) 1535-1542). However when the observation period was extended to after discharge from hospital, CRP detection proved to be predictive of future coronary events. Thus, Troponin and CRP detection facilitate risk evaluation at different stages of acute coronary syndrome and therefore the two tests together give more accurate predictions.
  • EP-A 1 076 240 describes and claims a method for determining levels of CRP using labelled PC, for example Eu labelled PC. Two types of sandwich assay methods using PC are described, namely antibody-PC (Method B) and PC-antibody (Method C). The methods described in EP-A 1 076 240 have the drawback involved in the use of immunological methods set out above.
  • the invention provides a method of detecting or determining C- reactive protein (CRP) in diverse animal species, which method comprises contacting a sample of a biological fluid from a human or non-human animal with a complex of a phosphorylated compound containing a nitrogen moiety and a label which is directly detectable in a non-immunological assay, the phosphoryl moiety and the nitrogen moiety being positioned relative to each other so as to permit binding to calcium-dependent ligand binding sites present on CRP.
  • CRP C- reactive protein
  • the method according to the invention enables one to carry out in a simple and rapid manner assays, some involving no signal amplification steps, which facilitate the non-immunological detection of CRP in a range of species including human, porcine and canine species. These assays enable the determination of the normal versus disease-state where elevated CRP levels are present.
  • the method is based solely on the interaction of CRP with the phosphorylated compound.
  • the complex has the formula
  • X is H or a group
  • Z is a linker molecule
  • n 0 or 1
  • W when present is a peptide or polypeptide
  • X' is a group S
  • S is a pyridoxal group, a tyrosinyl group or a group
  • Ri is H, (C ⁇ -C 4 )alkyl or a carboxy group
  • R 2 is H or (C r C 4 )alkyl
  • R 3 is H or (C r C 4 )alkyl
  • n 1 or 2
  • X' is a substituted or unsubstituted adenosyl or guanosyl group linked directly through the sugar moiety to a label or indirectly to said label by means of a peptide or polypeptide molecule, the label being directly detectable in a non-immunological assay.
  • the complex has the formula
  • Ri is H, (C ⁇ -C 4 )alkyl or a carboxy group
  • R 2 is H or (C C 4 )alkyl
  • R 3 is H or (C C 4 )alkyl
  • n 0 or 1 ;
  • n 1 or 2;
  • Z is a linker molecule;
  • W when present is a peptide or polypeptide
  • Y is a label which is directly detectable in a non- immunological assay.
  • a preferred compound is one wherein Ri and R 2 each represent a methyl group and n is 2 and Z is a cytidinyl phosphate moiety.
  • W is optional as indicated by the values for m, such that the label can be linked directly to the N-containing phosphorylated moiety.
  • W is present, it is preferably a protein selected from bovine serum albumin (BSA), casein, ovalbumin, thyroglobulin and keyhole limpet haemocyanin (KLH).
  • BSA bovine serum albumin
  • casein ovalbumin
  • thyroglobulin keyhole limpet haemocyanin
  • a preferred protein is BSA.
  • the label is selected from a microparticle, a gold label, an enzyme, a radio label and a lanthanide.
  • the label is a latex sphere or a gold label which is detectable in a nephelometric or turbidimetric assay as hereinafter described.
  • the label is the enzyme horseradish peroxidase which is detectable in an enzyme-linked sorbent assay as hereinafter described.
  • the biological fluid is from a canine, porcine or human animal.
  • biological fluid herein is meant a biological fluid as such including blood or a blood fraction such as plasma or a medium such as a buffer which is used to suspend or dilute the biological fluid.
  • CRP C-reactive protein
  • Ca calcium
  • PC binding offers a method to capture or immobilise CRP and facilitate subsequent detection.
  • Fig. 1A is a schematic representation of a turbidimetric assay system for CRP detection in accordance with the invention
  • Fig IB is a schematic representation of a CRP-ELSA in accordance with the invention.
  • Fig. 2 is a plot of ⁇ Absorbance 55 o nm versus CRP concentration ( ⁇ g/ml) for the turbidimetric assay system as described in Example 3 ;
  • Fig. 3 is a plot of Absorbance4 50 /620nm versus CRP concentration
  • Fig. 5 illustrates the detection of CRP in porcine plasma specimens ( ⁇ g/ml) exhibiting a range of CRP concentrations as described in Example 9;
  • Fig. 6 illustrates the variation in CRP levels in canine serum specimens obtained prior to and post-cruciate ligament surgery, as measured in the ELSA and an immunoassay utilising IgG [anti-canine CRP] as described in Example 9.
  • CDPC cytidine 5'-diphosphorylcholine
  • W carrier protein
  • BSA bovine serum albumin
  • the PC-BSA moiety can be either: (i) covalently coated directly onto microparticles and subsequently interacted with CRP from a number of species in a quantitative manner to form spectrophotometrically detectable complexes; or (ii) PC-BSA can be further modified by covalent attachment of horseradish peroxidase (HRP) to form PC-BSA-HRP which, in turn, can be used in a non- immunological enzyme-linked sorbent system (ELSA) to quantitatively detect CRP in biological fluids from various animal species.
  • HRP horseradish peroxidase
  • ELSA non- immunological enzyme-linked sorbent system
  • CDPC can be oxidised with sodium periodate to yield a reactive group capable of reacting with free amino (-NH 2 ) groups present on the carrier protein BSA. This bond can then be stabilised by the addition of sodium borohydride which catalyses the formation of a stable schiff base.
  • the PC-BSA conjugate still contains free -NH 2 groups which can be used for example to facilitate molecular coupling to either carboxyl-activated latex microparticles via carbodumide chemistry or to horseradish peroxidase via SMCC (4-(maleimidomethyl)cyclohexanecarboxylic acid N- hydroxysuccinimide ester) activation of PC-BSA and reaction with SATA (S-acetyl thioglycolic acid N-hydroxysuccinimide) -activated horseradish peroxidase.
  • SMCC 4-(maleimidomethyl)cyclohexanecarboxylic acid N- hydroxysuccinimide ester
  • a microparticle based assay is based on the principle that immobilised PC-BSA present on latex microspheres, typically of 200nm diameter, will form a stable non-covalent complex with free CRP present in a buffer or biological fluid.
  • This complex is spectrophotometically detectable by measuring the change in absorbance ( ⁇ A) at a particular wavelength, preferably 550nm, with time (typically 5 minutes) which in turn is dependent on the CRP amount or concentration ([CRP]) present in the reaction cuvette.
  • ⁇ A absorbance
  • time typically 5 minutes
  • CRP concentration
  • Fig. 1A immobilised PC (black circles) on latex particles binds free CRP in solution (indicated by dashed lines) to form insoluble complex.
  • the extent of complex formation, measured spectrophoto- metrically, is proportional to CRP concentration.
  • the enzyme-linked sorbent assay is a competitive assay format for the detection of free CRP in biological fluids and diluents, the function of which is also antibody-independent.
  • standards or specimens with or without CRP
  • PC-BSA-HRP phosphorylcholine-BSA-HRP
  • PC-BSA-HRP conjugate are jointly added to micro wells pre-coated with CRP. If there is no free CRP in the specimen (or zero standard) then the PC-BSA-HRP conjugate will preferentially bind to the CRP present on the microplate.
  • the pH of the reaction was maintained at 9.6 for 1 hour, using 5% sodium carbonate, after which reduction was performed overnight by the addition of 0.5M sodium borohydride (5ml). The pH was then lowered to pH 3.5 for 1 hour by adding IM formic acid and then neutralised with IM sodium hydroxide. Following this, the reaction mixture was dialysed exhaustively at 4°C in 50mM potassium phosphate, lmM EDTA, 150mM NaCl pH 7.8, with stirring, and then stored at -20°C until used for conjugation to horseradish peroxidase or coupling to microparticles.
  • a solution containing the following components was prepared: lOO ⁇ l 0.5M MES buffer pH 6.1, 546 ⁇ l of deionised water, lOO ⁇ l 10% (w/v) latex particles, 230 ⁇ l sulfo-NHS (50mg/ml) and 24 ⁇ l l-ethyl-3-(3- dimethylaminopropyl) carbodumide (ED AC) (lOmg/ml).
  • the components were mixed together on a shaker at room temperature for 30 minutes followed by centrifugation at 10,000g for 20 minutes. The supernatant was discarded.
  • the particles were resuspended by sonication in 1ml 50mM MES buffer pH 6.1 for washing and again centrifuged at 10,000g for 20 minutes. The supernatant was discarded and the particles were resuspended by sonication in 1ml of PC-BSA protein stock (1 mg/ml) in 0.1 M sodium borate buffer pH 8.5 and mixed at room temperature for 1 hour with shaking. Then, 50 ⁇ l 0.25M ethanolamine hydrochloride solution in 0.1 M sodium borate buffer was added and the solution mixed for a further 30 minutes at room temperature.
  • the activated particles were resuspended in 1ml of a lOmg/ml BSA in 0.1M sodium borate buffer (blocking buffer) by sonication and mixed for 30 minutes at room temperature. Finally, the particles (l%(w/v) suspension) were washed twice with 1ml aliquots of blocking buffer and finally resuspended in 1ml of 50mM Tris-HCl, 25mM NaCl, lOmM CaCl 2 pH 7.4, with sonication, and stored at 4°C until required for use.
  • Specimen diluent comprised 0.1M glycine, 90mM NaCl, 50mM calcium chloride, 0.04%(w/v) sodium azide pH 7.6; and
  • Particle diluent comprised 0.4%(w/v) PEG 6000 in sample diluent.
  • PC-BSA coated microparticles Prior to specimen dilution, PC-BSA coated microparticles were diluted to 0.03%(w/v) in particle diluent to give 'working reagent'.
  • Cobos Mira is a trade mark
  • autoanalyser pre-programmed to dispense either standards or specimens (5 ⁇ l each) along with specimen diluent (45 ⁇ l) and working reagent (450 ⁇ l) into reaction cuvettes, and brought to 37°C.
  • the reaction was initiated by addition of the working reagent and allowed to proceed at 37°C for 5 minutes.
  • PC-BSA (1 mg/ml) was modified with SMCC by adding 25mM SMCC to PC-BSA at a ratio of 7.5 ⁇ l 25mM SMCC per mg PC-BSA. The solution was mixed while making this addition and then incubated at room temperature for 30 minutes. The SMCC-activated PC-BSA (PC- BSA-SMCC) was then dialysed exhaustively against 50mM potassium phosphate, ImM EDTA, 150mM NaCl pH 6.8, with stirring, at 4°C.
  • HRP Horseradish peroxidase
  • Sulphydral groups were introduced into horseradish peroxidase by adding 0.05mg SATA er mg HRP (9 fold molar excess) and incubating at room temperature for lhour. Following this reaction, excess SATA was removed by exhaustive dialysis against 50 volumes of 50mM potassium phosphate, ImM EDTA, 150mM NaCl pH 6.8, with stirring, at 4°C. SATA-activated hoseradish peroxidase (SATA-HRP) was then stored at -20°C until required for conjugation to PC-BSA-SMCC.
  • SATA-HRP SATA-activated hoseradish peroxidase
  • SATA-HRP was deblocked (i.e., sulphydral groups exposed) by the addition of 500mM hydroxylamine (1/10 volume of SATA-HRP solution) and incubated, in the dark, at room temperature for 1 hour.
  • PC- BSA-SMCC was then added to deblocked HRP in the ratio of lmg PC- BSA-SMCC er 4.6mg of SATA-HRP, equivalent to a PC-BSA-SMCC: SATA-HRP molar ratio of 1 :7. This mixture was further incubated, in the dark, for 5 hours at room temperature.
  • NUNC Maxisorp is a trade mark flat bottomed plates (100 ⁇ l per well). The coated plates were incubated at 4°C for approximately 16 hours to allow antigen attachment and then washed 4 times with PBST and tapped dry. Microplates were blocked using a solution of 5%(w/v) Sucrose, 0.5%(w/v) BSA in PBS for 1 hour at 37° (200 ⁇ lper well). The blocking solution was then removed, the microplates tapped dry to remove any remaining blocking solution and incubated overnight at 37°C to dry. The coated microplates were then stored at 4°C until required for use.
  • Human CRP standards (range from 0-10 ⁇ g/ml) were prepared by serial dilution of a stock solution of CRP (24mg/ml) in 50mM MES, 25mM NaCl, lOmM CaCl 2 and 0.1% (v/v) Tween-20® pH 6.0 as follows: Human [CRP] Dilution
  • PC-BSA-HRP conjugate prepared in Example 6 and specimens for analysis were also diluted in 50mM MES, 25mM NaCl, lOmM CaCl 2 and 0.1% (v/v) Tween-20® pH 6.0.
  • 50 ⁇ l of standards or specimens diluted 1/40, 1/50 and 1/80 for porcine, human and canine CRP detection, respectively and 50 ⁇ l of the conjugate (diluted 1/8000) were added into microwells previously coated with 1 ⁇ g/ml human CRP (lOOng/well human CRP) as described in Example 7.
  • microwells were washed 4 times with 50mM Tris-HCl, 25mM NaCl, 1 OmM CaCl 2j 0.1% Tween-20® pH 7.4, with a 30 seconds soak stepper wash.
  • the microwells were tapped dry and lOO ⁇ l TMB were added per well followed by a 15 minute incubation.
  • the reaction was stopped by adding lOO ⁇ l IN H 2 S0 4 and the absorbance read at 450/630nm followed by calculation of specimen [CRP] from the standard curve depicted in Fig. 3.
  • the CRP-ELSA described herein was used to evaluate the levels of CRP in 23 human serum specimens in parallel with analysis of identical specimens by conventional nephelometric analysis (Behring Nephelometric Systems, Dade Behring Marburg GmbH). The results are shown in Fig. 4.
  • Figure 4 illustrates that all specimens tested gave near identical results using both systems with an observed correlation coefficient of 0.92.
  • CRP-ELSA for the detection of porcine and canine CRP, seven porcine plasma and six canine serum specimens were analysed.
  • Specimens 1-3 exhibited high levels of CRP (> 20 ⁇ g/ml) while specimens 4-7 all contained lower levels of CRP ( ⁇ 10 ⁇ g/ml).
  • This assay confirmed the utility of the CRP-ELSA described herein to detect variable levels of porcine CRP in clinical specimens.
  • Fig. 6 illustrates the results following the evaluation of canine serum CRP levels pre- and post-cruciate ligament surgery; wherein the open bars represent CRP concentration ( ⁇ g/ml) measured by ELSA in accordance with the invention and closed bars represent CRP concentration ( ⁇ g/ml) measured by other method.
  • X-axis labels numbers 1 and 2 refer to day 1 before surgery and immediately post-surgery, respectively.
  • Number 3 corresponds to 12 hours post-surgery while numbers 4, 5 and 6 relate to days 1, 15 and 48 post-surgery.
  • numbers 1 and 2 refer to day 1 before surgery and immediately post-surgery, respectively.
  • Number 3 corresponds to 12 hours post-surgery while numbers 4, 5 and 6 relate to days 1, 15 and 48 post-surgery.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé pour la détection et la détermination de la protéine C réactive (CRP) chez diverses espèces animales. Ce procédé consiste à mettre en contact un échantillon d'un liquide biologique prélevé chez un animal humain ou non avec un complexe d'un composé phosphorylé contenant une fraction azote et un marqueur directement détectable dans un dosage non immunologique. La fraction phosphoryle et la fraction azote sont positionnées l'une par rapport à l'autre de façon à permettre la liaison aux sites de liaison du ligand dépendant du calcium, présents sur la CRP. Ce procédé permet d'effectuer des dosages de façon simple et rapide, certains ne nécessitant pas d'étapes d'amplification de signal, ce qui facilite la détection non immunologique de la CRP chez diverses espèces, notamment humaine, porcine et canine. Ces dosages permettent la détermination d'un état normal par rapport à un état pathologique caractérisé par des taux élevés de CRP.
PCT/IE2001/000124 2001-09-27 2001-09-27 Dosages non immunologiques pour la detection et la determination de la proteine c reactive WO2003036297A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/IE2001/000124 WO2003036297A1 (fr) 2001-09-27 2001-09-27 Dosages non immunologiques pour la detection et la determination de la proteine c reactive

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/IE2001/000124 WO2003036297A1 (fr) 2001-09-27 2001-09-27 Dosages non immunologiques pour la detection et la determination de la proteine c reactive

Publications (1)

Publication Number Publication Date
WO2003036297A1 true WO2003036297A1 (fr) 2003-05-01

Family

ID=11042207

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IE2001/000124 WO2003036297A1 (fr) 2001-09-27 2001-09-27 Dosages non immunologiques pour la detection et la determination de la proteine c reactive

Country Status (1)

Country Link
WO (1) WO2003036297A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104630151A (zh) * 2015-01-19 2015-05-20 中国农业大学 一种检测猪c反应蛋白的单克隆抗体
WO2016060096A1 (fr) * 2014-10-15 2016-04-21 日油株式会社 Composé contenant un groupe phosphorylcholine et complexe de phosphorylcholine
CN111675764A (zh) * 2020-05-13 2020-09-18 浙江正熙生物医药有限公司 一种藻红蛋白免疫荧光探针及其标记蛋白的方法

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS62258390A (ja) * 1986-05-02 1987-11-10 Oriental Yeast Co Ltd 新規ホスホリルコリン誘導体の製造法
JPS62258399A (ja) * 1986-05-02 1987-11-10 Oriental Yeast Co Ltd Ｃ−反応性蛋白質の精製方法
JPS62259063A (ja) * 1986-05-02 1987-11-11 Oriental Yeast Co Ltd Ｃ−反応性蛋白質の定量方法
GB2217335A (en) * 1988-04-22 1989-10-25 Axis Research Compositions for isolation and immobilisation of C-reactive protein in body liquids
GB2217840A (en) * 1988-04-22 1989-11-01 Axis Research Compositions for detection and/or quantification of an isolated acute phase protein in body liquids
EP1076240A1 (fr) * 1999-03-03 2001-02-14 Oriental Yeast Co., Ltd. Methode de dosage de crp a l'aide de phosphorylcholine
EP1130396A1 (fr) * 2000-02-29 2001-09-05 Kyowa Medex Co., Ltd. Méthode de détermination et réactif de détermination de la protéine C réactive

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS62258390A (ja) * 1986-05-02 1987-11-10 Oriental Yeast Co Ltd 新規ホスホリルコリン誘導体の製造法
JPS62258399A (ja) * 1986-05-02 1987-11-10 Oriental Yeast Co Ltd Ｃ−反応性蛋白質の精製方法
JPS62259063A (ja) * 1986-05-02 1987-11-11 Oriental Yeast Co Ltd Ｃ−反応性蛋白質の定量方法
GB2217335A (en) * 1988-04-22 1989-10-25 Axis Research Compositions for isolation and immobilisation of C-reactive protein in body liquids
GB2217840A (en) * 1988-04-22 1989-11-01 Axis Research Compositions for detection and/or quantification of an isolated acute phase protein in body liquids
EP1076240A1 (fr) * 1999-03-03 2001-02-14 Oriental Yeast Co., Ltd. Methode de dosage de crp a l'aide de phosphorylcholine
EP1130396A1 (fr) * 2000-02-29 2001-09-05 Kyowa Medex Co., Ltd. Méthode de détermination et réactif de détermination de la protéine C réactive

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PATENT ABSTRACTS OF JAPAN vol. 012, no. 138 (P - 695) 27 April 1988 (1988-04-27) *
PATENT ABSTRACTS OF JAPAN vol. 012, no. 143 (C - 492) 30 April 1988 (1988-04-30) *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016060096A1 (fr) * 2014-10-15 2016-04-21 日油株式会社 Composé contenant un groupe phosphorylcholine et complexe de phosphorylcholine
US9850266B2 (en) 2014-10-15 2017-12-26 Nof Corporation Phosphorylcholine group-containing compound and phosphorylcholine complex
CN104630151A (zh) * 2015-01-19 2015-05-20 中国农业大学 一种检测猪c反应蛋白的单克隆抗体
CN111675764A (zh) * 2020-05-13 2020-09-18 浙江正熙生物医药有限公司 一种藻红蛋白免疫荧光探针及其标记蛋白的方法
CN111675764B (zh) * 2020-05-13 2023-09-08 浙江正熙生物技术有限公司 一种藻红蛋白免疫荧光探针及其标记蛋白的方法

Similar Documents

Publication Publication Date Title
JP6441407B2 (ja) 非特異的結合を減少させるためのイムノアッセイ方法及び試薬
JP2726793B2 (ja) 免疫アッセイ
AU2018374469B2 (en) Target interference suppressed anti-drug antibody assay
JP7398366B2 (ja) 対称性ジメチルアルギニンの検出
US5362655A (en) Process for the determination of a specifically bindable substance
WO2018051965A1 (fr) Procédé de dosage de troponine cardiaque et réactif de dosage
KR20090034999A (ko) 응집 저해 측정법 및 응집 저해 측정용 시약
US5814461A (en) Method for the determination of anti-TSH receptor autoantibodies
JPH1078435A (ja) 被検物質の免疫化学的測定における感度の上昇
Deegan et al. Quantitative detection of C-reactive protein using phosphocholine-labelled enzyme or microspheres
JP2002296281A (ja) 改良された均一系イムノアッセイ法
WO2021250467A2 (fr) Détection d'anticorps contre le sars-cov-2
US8900882B2 (en) Method of assaying complex and kit to be used therefor
WO2003036297A1 (fr) Dosages non immunologiques pour la detection et la determination de la proteine c reactive
EP1239285A1 (fr) Méthode améliorée pour un procédé d'essai immunologique homogène
US20190339264A1 (en) Immunoassay controls and the use thereof
JPH04236353A (ja) 抗体の測定方法
KR940008091B1 (ko) 리간드의 면역학적 검출방법
Losso et al. Development of a particle concentration fluorescence immunoassay for the quantitative determination of IgG in bovine milk
JPH11295313A (ja) 抗体または抗体断片の重合体とその利用
CA2246894C (fr) Surfaces de liaison pour ligands
JP3768165B2 (ja) 抗カルパスタチン抗体の測定法及び測定キット
JP2024046503A (ja) 細胞外小胞に含まれるアルカリフォスファターゼの活性を測定する方法、キャリブレータ及び結合体
JP2004271502A (ja) 酸化ｌｄｌ−ｃｒｐ複合体の測定方法及び測定キット
CN117886803A (zh) N-乙酰神经氨酸半抗原、n-乙酰神经氨酸半抗原人工抗原及其制备方法

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PH PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP