CN108593641B

CN108593641B - Kit and method for quantitatively detecting substance to be detected in whole blood sample

Info

Publication number: CN108593641B
Application number: CN201810311589.5A
Authority: CN
Inventors: 周永杨; 周剑青; 霍利仁
Original assignee: Urit Medical Electronic Co Ltd
Current assignee: Urit Medical Electronic Co Ltd
Priority date: 2018-04-09
Filing date: 2018-04-09
Publication date: 2021-12-03
Anticipated expiration: 2038-04-09
Also published as: CN108593641A

Abstract

The present application relates to the field of immunological assays. Specifically, the present application relates to a kit for quantitatively detecting a substance to be detected in a whole blood sample, which comprises insoluble carrier particles, an antibody specifically binding to the substance to be detected, and a first reagent composition comprising a betaine-type surfactant. Further, the present application relates to a method for detecting the presence or amount of a substance to be tested in a whole blood sample, which comprises the steps of contacting a non-hemolyzed whole blood sample with an insoluble carrier particle coated with an antibody specifically binding to the substance to be tested in the first reagent composition and measuring a change in turbidity of the reaction system.

Description

Kit and method for quantitatively detecting substance to be detected in whole blood sample

Technical Field

The present application relates to the field of immunological assays. Specifically, the application relates to a kit for quantitatively detecting a substance to be detected in a whole blood sample. In addition, the present application relates to a method for detecting the presence or amount of a substance to be tested in a whole blood sample.

Background

Human C-reactive protein (CRP) is a pentameric protein, non-covalently linked by five identical subunits, with a relative molecular mass of 115-140 kD. The half-life of CRP is about 15h, the concentration of normal people is very low, but the CRP begins to rise 6-8h after tissue injury and acute infection occur, the peak value of the CRP reaches 24-48h and can reach hundreds of times or even thousands of times of the normal value, the rising amplitude is in direct proportion to the infection degree, the concentration is rapidly reduced after the inflammation is cured, and the normal level can be recovered one day. The continuous increase of CRP indicates that the body has chronic inflammation or autoimmune disease, and the CRP is not increased generally when infected by virus, and the change is not influenced by individual difference of patients, body state and therapeutic drugs. CRP has good guiding function for differential diagnosis of bacterial or viral infection, monitoring of activity of diseases and monitoring of curative effect.

In recent years, with intensive research on clinical significance of CRP, the application range of CRP in clinic is more and more extensive. At present, samples for clinical detection are generally serum or plasma, whole blood samples need to be pre-separated into red blood cells and then tested, special separation equipment is needed, operation steps are complex, time and labor are wasted, the required samples are large in size, particularly, blood collection for children, newborns, large-area burn patients and the like is difficult, and the requirement of clinical rapid diagnosis cannot be met. Therefore, the reagent for rapidly detecting CRP by using whole blood as a sample is extremely urgent and important.

The CRP assay kit currently available on the market is a double reagent consisting of reaction buffer 1(R1) and a latex particle reagent (R2) coupled with CRP antibody. In the detection of a sample by using the kit, generally, the whole blood sample is hemolyzed by adding R1 in the first step, and then the whole blood sample is reacted by adding R2 in the second step, so that the detection speed is influenced, the reagent cost is increased, and the antibody in R2 can be inactivated due to the change of the liquid environment when the two are premixed together, so that the stability of the reagent is influenced.

Disclosure of Invention

The inventor has found, surprisingly, through a large number of experimental studies: in the quantitative determination of a substance to be measured (for example, C-reactive protein) in a whole blood sample based on immunoturbidimetry, a non-hemolyzed whole blood sample can be directly contacted with an antibody-sensitized latex particle by using a specific reagent composition, thereby detecting a change in turbidity thereof. Based on this finding, the present inventors have developed a novel quantitative detection kit and a detection method for a substance to be detected in a whole blood sample.

Reagent kit

Accordingly, in one aspect, the present invention provides a kit comprising:

(a) insoluble carrier particles;

(b) an antibody that specifically binds to the test substance; and

(c) a first reagent composition comprising a betaine-type surfactant.

In certain preferred embodiments, the kit is used for immunoturbidimetric determination of the presence or amount of a test substance in a blood sample. In certain preferred embodiments, the blood sample is selected from whole blood, plasma, or serum. In certain preferred embodiments, the blood sample is anticoagulated whole blood and is not hemolyzed.

In certain preferred embodiments, the test substance is a protein in a plasma fraction, such as C-reactive protein and the like. In certain exemplary embodiments, the test agent is C-reactive protein (CRP).

In certain preferred embodiments, the betaine-type surfactant is selected from the group consisting of alkyl betaines, alkyl amidobetaines, sulfopropyl betaines, hydroxysulfopropyl betaines, phospholipidbetaines, and any combination thereof. In certain preferred embodiments, the first reagent composition comprises 3-sulfopropyl-hexadecyldimethyl betaine. In certain preferred embodiments, the betaine-type surfactant is present in an amount of 0.01-4% (w/v). In certain preferred embodiments, the betaine-type surfactant is present in an amount of 0.05-0.5% (w/v).

In certain preferred embodiments, the kit further comprises a second reagent composition comprising a blocking agent. In certain preferred embodiments, the blocking agent is N6-PEG.

In certain preferred embodiments, the first reagent composition further comprises one or more reagents selected from the group consisting of: buffers, preservatives, suspending agents, stabilizers and coagulants.

In certain preferred embodiments, the buffer is selected from the group consisting of glycine buffer, MES buffer, MOPSO buffer, Tris buffer, phosphate buffer, borate buffer, and any combination thereof. In certain preferred embodiments, the buffer is present in an amount of 0.02 to 0.1 mol/L.

In certain preferred embodiments, the preservative is selected from the group consisting of sodium azide, sorbate salts, benzoic acid and its salts, sodium nitrite, and Prolin-300. In certain preferred embodiments, the preservative is present in an amount of 0.5 to 1.5 g/L.

In certain preferred embodiments, the suspending agent is selected from the group consisting of glycerol, ethylene glycol, mannitol, and any combination thereof. In certain preferred embodiments, the suspending agent is present in an amount of 1-30% (v/v).

In certain preferred embodiments, the stabilizing agent is sodium chloride, magnesium chloride, disodium edetate, bovine serum albumin, glycine, gelatin, and any combination thereof. In certain preferred embodiments, the sodium chloride or magnesium chloride is present in an amount of 0.05-1.5% (w/v), the disodium edetate is present in an amount of 0.01-1% (w/v), and the bovine serum albumin or gelatin is present in an amount of 0.1-3% (w/v).

In certain preferred embodiments, the coagulant is selected from the group consisting of polyethylene glycol 6000, polyethylene glycol 8000, polyethylene glycol 10000, polyethylene glycol 20000, dextran sulfate, and any combination thereof. In certain preferred embodiments, the coagulant is present in an amount of 0.5-3% (w/v).

In certain preferred embodiments, the first reagent composition consists of: betaine type surfactant, antiseptic, suspending agent, coagulant, buffer solution and water.

In certain exemplary embodiments, the first reagent composition consists of: 3-sulfopropylhexadecylbetaine, sodium azide, mannitol, polyethylene glycol (e.g., polyethylene glycol 6000), glycine, and balance water (e.g., deionized water, distilled water, etc.).

In certain exemplary embodiments, the first reagent composition consists of: 0.05% (w/v) 3-sulfopropylhexadecylbetaine, 1g/L sodium azide, 0.01M mannitol, 0.5% (w/v) polyethylene glycol 6000, 0.01M glycine, and the balance water (e.g., deionized water, distilled water, etc.).

In the present invention, the insoluble carrier particles include any carrier particles known to be useful in immunoturbidimetry (enhanced turbidimetry), such as latex particles or polylactic acid particles. In certain preferred embodiments, the insoluble carrier particles are latex particles. The material of the latex particles is not particularly limited, and non-limiting examples thereof include polystyrene, styrene-butadiene copolymer, styrene-acrylate copolymer, styrene-maleic acid copolymer, polyethyleneimine, polyacrylic acid, polymethacrylic acid, polymethylmethacrylate, and the like. In certain preferred embodiments, the insoluble support particles are polystyrene particles.

In certain preferred embodiments, the second reagent composition further comprises a reagent for coating the antibody on the surface of the insoluble carrier particle, and optionally a coating buffer (e.g., carbonate buffer, phosphate buffer, Tris-HCL buffer, borate buffer, or the like).

In the present invention, the antibody may be coated on the surface of the insoluble carrier particle by various techniques known to those skilled in the art, such as physical adsorption or covalent coupling by surface functionalization (e.g., carboxyl, amino, hydroxyl, hydrazide, or thiol). Detailed teachings of Covalent Coupling can be found, for example, in TechNote 205, rev.003, for example March,2002, "compatible Coupling" (incorporated herein by reference), which can be downloaded from the web pages of Bangs Laboratories, Inc. The skilled person is familiar with how to select suitable reagents for coating an antibody on the surface of insoluble carrier particles, depending on the functional groups modified on the surface of said insoluble carrier particles.

Thus, in certain preferred embodiments, the insoluble carrier particles have carboxyl, amino, hydroxyl, hydrazide, or thiol modifications on the surface. In certain exemplary embodiments, the insoluble carrier particles have a carboxyl modification on the surface. In such embodiments, the antibody may be coated on the surface of the insoluble carrier particle by a coupling reaction between amino and carboxyl groups. Thus, in certain exemplary embodiments, the second reagent composition comprises EDC or a salt thereof (e.g., EDCI) and NHS.

In certain preferred embodiments, the surface of the insoluble carrier particles is coated with an antibody that specifically binds to the test substance.

In certain preferred embodiments, the surface of the insoluble carrier particles is coated with an antibody that specifically binds to the test substance, and N6-PEG.

In certain exemplary embodiments, the test agent is C-reactive protein (CRP). In such embodiments, the kit comprises an antibody that specifically binds CRP.

In certain preferred embodiments, the antibody is a monoclonal antibody or a polyclonal antibody. In certain preferred embodiments, the antibody is a polyclonal antibody.

In certain preferred embodiments, the kit further comprises one or more reagents or devices selected from the group consisting of: standards (e.g., a series of samples containing different known amounts of a substance to be tested); a positive control sample (e.g., a sample containing a known amount of a test substance); a negative control sample (e.g., a sample that does not contain the test substance); anticoagulants (e.g., heparin); and, a blood collection device (e.g., a pyrogen-free evacuated blood collection tube).

In certain preferred embodiments, the components comprised by the kit of the invention, as described above, are provided separately.

In certain preferred embodiments, the insoluble carrier particle, the antibody that specifically binds to the substance to be tested, the first reagent composition, and the second reagent composition are provided separately.

In certain preferred embodiments, the insoluble carrier particles are provided as the same component as the first reagent composition. In such embodiments, the surface of the insoluble carrier particle is coated with an antibody that specifically binds to the test substance; alternatively, the surface of the insoluble carrier particle is coated with an antibody specifically binding to the substance to be detected, and N6-PEG. In certain preferred embodiments, the insoluble carrier particles are provided in combination with the first reagent composition in a suspended form.

Detection method

In another aspect, the present invention provides an immunoturbidimetric method for determining the presence or amount of a test substance in a sample of unhemolyzed whole blood, comprising the steps of:

(1) contacting the non-hemolyzed whole blood sample with insoluble carrier particles in a first reagent composition under conditions permissive for antigen-antibody complex formation;

(2) measuring the turbidity change of the reaction system in the step (1);

(3) comparing the turbidity change obtained in the step (2) with a standard curve representing the relation between the known amount of the substance to be detected and the turbidity change, and obtaining the content of the substance to be detected;

wherein the surface of the insoluble carrier is coated with an antibody which specifically binds to the substance to be detected; and, the first reagent composition comprises a betaine-type surfactant.

In certain preferred embodiments, the whole blood sample is anticoagulated whole blood.

In certain preferred embodiments, the non-hemolyzed whole blood sample contains the test substance. In certain preferred embodiments, the non-hemolyzed whole blood sample comprises CRP.

In certain preferred embodiments, the methods of the invention are used for non-diagnostic purposes.

In certain preferred embodiments, the betaine-type surfactant is selected from one or more of alkyl betaines, alkyl amidobetaines, sulfopropyl betaines, hydroxysulfopropyl betaines, phospholipidbetaines. In certain preferred embodiments, the first reagent composition comprises 3-sulfopropyl-hexadecyldimethyl betaine. In certain preferred embodiments, the betaine-type surfactant is present in an amount of 0.01-4% (w/v).

In certain preferred embodiments, in step (1), the insoluble carrier particles are surface coated with N6-PEG.

In certain preferred embodiments, the insoluble carrier particles are contained in the reaction system of step (1) in an amount of 2 to 5 g/L.

In certain preferred embodiments, the insoluble carrier particles are latex particles. The material of the latex particles is not particularly limited, and non-limiting examples thereof include polystyrene, styrene-butadiene copolymer, styrene-acrylate copolymer, styrene-maleic acid copolymer, polyethyleneimine, polyacrylic acid, polymethacrylic acid, polymethylmethacrylate, and the like. In certain preferred embodiments, the insoluble support particles are polystyrene particles.

In certain preferred embodiments, in step (1), the antibody coated on the surface of the insoluble carrier particle is capable of immunoreacting with a test substance, thereby aggregating the insoluble carrier particle in a reaction system to cause an agglutination reaction.

In the present invention, a method for measuring the change in turbidity of the reaction system of the step (1) is well known to those skilled in the art. For example, the change in absorbance or scattered light at a predetermined wavelength within a certain time (for example, 0 to 5 minutes, 0 to 2 minutes, 5 to 120 seconds, or 5 to 60 seconds) after the start of the reaction in step (1) can be measured by an optical method as the change in turbidity.

Thus, in certain preferred embodiments, in step (2), after the reaction of step (1) has begun: (a) measuring the absorbance of the reaction system at a prescribed wavelength for 2 times at appropriate time intervals, and taking the difference as the amount of change in absorbance (i.e., change in turbidity); or, (b) continuously measuring the absorbance of the reaction system at a predetermined wavelength, and taking the absorbance change rate per unit time as the change amount of absorbance (that is, change in turbidity).

In certain preferred embodiments, in step (2), the absorbance of the reaction system at a prescribed wavelength is measured 2 times within 0 to 120 seconds (e.g., 5 to 120 seconds, e.g., 5 to 60 seconds) after the start of the reaction of step (1), and the difference is taken as the amount of change in absorbance (i.e., change in turbidity). In certain preferred embodiments, in step (2), the absorbance of the reaction system at a prescribed wavelength is measured 2 times at 6 th and 60 th seconds, respectively, after the start of the reaction of step (1), and the difference is taken as the amount of change in absorbance (i.e., change in turbidity).

In certain preferred embodiments, the defined wavelength is 800-. In certain exemplary embodiments, the prescribed wavelength is 850 nm.

In certain exemplary embodiments, in step (2), the absorbance of the reaction system at 850nm is measured 2 times at 6 th and 60 th seconds after the start of the reaction of step (1), respectively, and the difference is taken as the amount of change in absorbance (i.e., change in turbidity).

In certain preferred embodiments, prior to step (1), there is further included the step of blocking the insoluble carrier particles with N6-PEG. Thus, in certain preferred embodiments, in step (1), the insoluble carrier particles are further coated with N6-PEG.

In certain preferred embodiments, prior to step (1), further comprising one or more of the following steps: (a) obtaining a whole blood sample from a subject using a blood collection device; (b) the blood collection device or the whole blood sample is treated with an anticoagulant (e.g., heparin).

Detection application

In another aspect, the present invention relates to the use of a betaine-type surfactant in the preparation of a kit for determining the presence or amount of a test substance in a non-hemolyzed whole blood sample by immunoturbidimetry, wherein the kit comprises:

(a) insoluble carrier particles;

(b) an antibody that specifically binds to the test substance; and

(c) a first reagent composition comprising a betaine-type surfactant.

In certain preferred embodiments, the present invention relates to the use of a betaine-type surfactant, insoluble carrier particles and an antibody that specifically binds to the test substance for the preparation of a kit for determining the presence or amount of the test substance in a non-hemolyzed whole blood sample by immunoturbidimetry.

In certain preferred embodiments, the betaine-type surfactant is selected from the group consisting of alkyl betaines, alkyl amidobetaines, sulfopropyl betaines, hydroxysulfopropyl betaines, phospholipidbetaines, and any combination thereof. In certain preferred embodiments, the betaine-type surfactant is 3-sulfopropyl-hexadecyldimethyl betaine. In certain preferred embodiments, the betaine-type surfactant is present in an amount of 0.01-4% (w/v). In certain preferred embodiments, the betaine-type surfactant is present in an amount of 0.05-0.5% (w/v).

In certain preferred embodiments, the present invention relates to the use of a betaine-type surfactant, N6-PEG, insoluble carrier particles, and an antibody that specifically binds to the test substance, for the preparation of a kit for determining the presence or amount of the test substance in a sample of unhemolyzed whole blood by immunoturbidimetry.

In certain preferred embodiments, the insoluble carrier particles have carboxyl, amino, hydroxyl, hydrazide, or thiol modifications on the surface. In certain exemplary embodiments, the insoluble carrier particles have a carboxyl modification on the surface. In such embodiments, the antibody may be coated on the surface of the insoluble carrier particle by a coupling reaction between amino and carboxyl groups. Thus, in certain exemplary embodiments, the second reagent composition comprises EDC or a salt thereof (e.g., EDCI) and NHS.

In certain preferred embodiments, the kit determines the presence or amount of the test substance in a sample of unhemolyzed whole blood by a method comprising the steps of:

(1) contacting the non-hemolyzed whole blood sample with the insoluble carrier particle in the first reagent composition under conditions that allow for antigen-antibody complex formation;

(2) measuring the turbidity change of the reaction system in the step (1);

wherein the surface of the insoluble carrier is coated with an antibody that specifically binds to the substance to be detected.

In certain preferred embodiments, the non-hemolyzed whole blood sample comprises CRP.

In certain preferred embodiments, in step (2), the change in absorbance or scattered light at a prescribed wavelength within a certain time (for example, 0 to 5 minutes, 0 to 2 minutes, 5 to 120 seconds, or 5 to 60 seconds) after the start of the reaction of step (1) is measured by an optical method as the change in turbidity.

In certain preferred embodiments, in step (2), after the reaction of step (1) has begun: (a) measuring the absorbance of the reaction system at a prescribed wavelength for 2 times at appropriate time intervals, and taking the difference as the amount of change in absorbance (i.e., change in turbidity); or, (b) continuously measuring the absorbance of the reaction system at a predetermined wavelength, and taking the absorbance change rate per unit time as the change amount of absorbance (that is, change in turbidity).

Preparation method

In another aspect, the present invention provides a method for preparing antibody-coated insoluble carrier particles, comprising the steps of:

(1) coating the antibody on the surface of insoluble carrier particles;

(2) incubating the blocking agent with the product of step (1), said blocking agent being N6-PEG.

In certain preferred embodiments, in step (1), the antibody is coated on the surface of an insoluble support by covalent coupling. In such embodiments, the insoluble support particles described in step (1) have a functional group (e.g., carboxyl, amino, hydroxyl, hydrazide, or thiol) modification on the surface.

In certain preferred embodiments, in step (1), the antibody is coated on the surface of the insoluble carrier particle by a coupling reaction between an amino group and a carboxyl group. In such embodiments, the insoluble carrier particles described in step (1) carry a carboxyl modification on their surface. Thus, in certain exemplary embodiments, in step (1), the antibody is contacted with the insoluble support in the presence of EDC or a salt thereof (e.g., EDCI) and NHS. In certain exemplary embodiments, in step (1), the antibody is contacted with the insoluble support in a buffer comprising EDC or a salt thereof (e.g., EDCI) and NHS. In certain preferred embodiments, the buffer is selected from the group consisting of glycine buffer, MES buffer, MOPSO buffer, Tris buffer, phosphate buffer, and borate buffer, and any combination thereof.

In certain preferred embodiments, in step (2), the blocking agent is present in an amount of 0.4% to 2% (w/v). In certain preferred embodiments, the blocking agent is present in an amount of 1% (w/v).

In certain preferred embodiments, after step (2), there is further included a step of washing the product of step (2) to remove unreacted materials. In certain preferred embodiments, the product of step (2) is washed with a buffer. In certain exemplary embodiments, the buffer is selected from the group consisting of phosphate buffer, MES buffer, Tris buffer, and the like.

Abbreviations and definitions of terms

The following abbreviations are used herein:

EDC 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC)

EDCI 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI)

NHS N-hydroxysuccinimide

MES 2- (N-morpholine) ethanesulfonic acid

MOPSO 3- (N-morpholinyl) 2-hydroxypropanesulfonic acid

Tris Tris hydroxymethyl aminomethane

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, virological, biochemical, immunological laboratory procedures used herein are all routine procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

As used herein, the term "immunoturbidimetry" refers to a method of detecting or quantifying an antigen or an antibody in a test sample based on a change in turbidity (for example, optical properties such as absorbance or scattered light) of a reaction system caused by an antigen-antibody reaction, and belongs to one of immunological detection methods. Generally, in order to improve the sensitivity of detection, an antibody or antigen capable of specifically binding a target antigen or a target antibody in a test sample is immobilized on an insoluble carrier particle (e.g., latex particle) to form a sensitized particle; the method, also called Particle-enhanced turbidimetric immunoassay (PETIA), detects or quantifies a test substance based on a change in optical properties caused by the agglutination of the sensitized particles due to the antigen-antibody reaction; in particular, when Latex particles are used as insoluble solid particles, the method is also referred to as Latex-enhanced turbidimetric immunoassay (LIA). In the present invention, the expression "immunoturbidimetry" means immunoturbidimetry using insoluble carrier particles.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules (i.e., a binding molecule and a target molecule), such as a reaction between an antibody and an antigen against which it is directed. Binding affinity between two molecules may be represented by K_DThe value describes. K_DThe value refers to the dissociation constant derived from the ratio of kd (the dissociation rate of a particular binding molecule-target molecule interaction; also known as koff) to ka (the association rate of a particular binding molecule-target molecule interaction; also known as kon), or kd/ka expressed as molarity (M). K_DThe smaller the value, the more tightly bound the two molecules and the higher the affinity. In certain embodiments, an antibody that specifically binds to (or is specific for) an antigen means that the antibody is present in an amount less than about 10^-5M, e.g. less than about 10^-6M、10^-7M、10^-8M、10^-9M or 10^-10M or less affinity (K)_D) Binding the antigen. K_DValues can be determined by methods well known in the art, for example, in a BIACORE instrument using Surface Plasmon Resonance (SPR).

As used herein, the expression "whole blood sample is not hemolyzed" means that the whole blood sample has not been subjected to any hemolysis treatment after being obtained from a subject.

As used herein, the term "Betaine type surfactant" refers to an amphoteric surfactant composed of a quaternary ammonium salt type cation moiety and a carboxylate type anion moiety. Examples include, but are not limited to, alkyl betaines, alkyl amide betaines, sulfopropyl betaines, hydroxysulfopropyl betaines, phosphoester betaines, and the like.

As used herein, the term "N6-PEG" refers to a "Methoxy-polyethylene glycol-pentaethylenehexamine (α -Methoxy-poly (ethylene glycol) -pentaethylenehexamine)" polymer having 6 amino groups attached to a single end of a polyethylene glycol segment and having the formula shown below, wherein m is 0, 1, 2, 3, or 4, and N is an integer of from 10 to about 200 (preferably from about 100 to about 200, for example from about 100 to about 150, for example from about 100 to about 120). For a detailed teaching of N6-PEG see, for example, Furusho H et al Chemistry of materials 2009,21, 3526-3535. N6-PEG is available from JSR Life Sciences, Japan under the trade designation CE 510.

As used herein, the term "blocking agent" refers to any substance that is capable of reducing non-specific interactions, such as non-specific antibody binding. Such materials are well known to those skilled in the art and examples include, but are not limited to, gelatin, bovine serum albumin, ovalbumin, casein, and skim milk, among others.

As used herein, the term "procoagulant" refers to a substance capable of accelerating the formation of an antigen-antibody complex. Such substances are well known to those skilled in the art, and examples thereof include, but are not limited to, water-soluble polymers such as polyethylene glycol, polyvinyl alcohol, dextran, sodium chondroitin sulfate, and the like.

As used herein, the term "suspending agent" refers to a substance that increases the viscosity of the dispersion medium to reduce the settling rate of the microparticles. In the present invention, the suspending agent is preferably a low molecular suspending agent such as glycerin, ethylene glycol, mannitol, and the like.

As used herein, the term "stabilizer" refers to a substance that controls or inhibits the self-coagulation of insoluble carrier particles (e.g., latex particles), such substances being well known to those skilled in the art, examples of which include, but are not limited to, sodium chloride, magnesium chloride, disodium edetate, bovine serum albumin, glycine, gelatin, and the like.

As used herein, the term "buffer" refers to a solution that is capable of preventing a significant change in pH by the action of its acid-base pairing components. Such materials are well known to those skilled in the art and may be found, for example, in Buffers.A. Guide for the Preparation and Use of Buffers in Biological Systems, Gueffroy, D.E., ed.Calbiochem Corporation (1975). Non-limiting examples of buffers include MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate, ammonium salts, and the like.

As used herein, the term "positive control sample" refers to a sample containing a known amount of C-reactive protein. In certain embodiments, the amount of test substance (e.g., C-reactive protein) in the test sample can be calculated by comparing the test results of the test sample with the test results of the positive control sample, such methods being well known in the art, e.g., by constructing a standard curve by testing different concentrations of the positive control sample.

As used herein, the term "anticoagulant" refers to a substance that is capable of preventing the coagulation of blood. Such materials are well known to those skilled in the art, and examples include, but are not limited to, heparin, EDTA, oxalate (e.g., sodium, potassium or ammonium oxalate), sodium poncirate, and the like.

Advantageous effects of the invention

The CRP assay kit currently available on the market is a double reagent consisting of reaction buffer 1(R1) and a latex particle reagent (R2) coupled with CRP antibody. In practical application, the whole blood sample is required to be hemolyzed by adding R1 in the first step, and then the reaction is carried out by adding R2 in the second step. Thus, the detection speed is influenced, and the reagent cost is increased; and the two are premixed together, and the antibody in the R2 can be inactivated due to the change of liquid environment, so that the stability of the reagent is influenced.

The invention provides a quantitative detection kit for a substance to be detected in a whole blood sample containing a specific reagent composition and a quantitative detection method for the substance to be detected in the whole blood sample established based on the reagent composition. Compared with the prior art, the technical scheme of the invention can directly detect the non-hemolytic whole blood sample, namely the whole blood sample can be directly contacted with the latex particles sensitized by the antibody without carrying out prior hemolytic treatment on the whole blood sample, so that the turbidity change of the whole blood sample is detected, the reagent cost is greatly reduced, the operation is simpler, the detection speed is greatly improved, and the result is accurate and reliable. Therefore, the technical scheme of the invention is particularly suitable for quickly diagnosing the infection degree of the patient and evaluating the prognosis.

Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention and do not limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments.

Drawings

FIG. 1 shows a linear regression analysis of the theoretical concentration versus the actual measured concentration of the CRP calibrator in example 1.

FIG. 2 shows a CRP calibration curve for N6-PEG with BSA as a blocking agent in example 2.

FIG. 3 shows the detection curves for CRP calibrators obtained in example 3 using different hemolysing agents.

FIG. 4 shows a regression analysis of the assay results obtained by the whole blood CRP assay of the invention and the conventional serum CRP assay of example 4.

Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

Unless otherwise indicated, the molecular biological experimental methods and immunoassay methods used in the present invention are essentially described by reference to j.sambrook et al, molecular cloning: a laboratory manual, 2 nd edition, cold spring harbor laboratory Press, 1989, and F.M. Ausubel et al, eds. molecular biology laboratory Manual, 3 rd edition, John Wiley & Sons, Inc., 1995; the use of restriction enzymes follows the conditions recommended by the product manufacturer. The examples are given by way of illustration and are not intended to limit the scope of the invention as claimed.

Preparation example 1: preparation of whole blood C reactive protein detection kit

In the present preparation example, latex reagents (JSR Life Sciences) having an average diameter of 68nm and 198nm were sensitized with goat anti-human CRP antibodies (Guilin Enmet Biotechnology Co., Ltd.), respectively, and then the reagents were mixed in a certain ratio to obtain a reaction reagent. The method comprises the following specific steps:

adding 3mL of MES buffer (pH 5.8) to 1mL of carboxylated latex particles (taking the example that the average diameter of the latex particles is 68nm and the solid content is 5%), adding 10mg/mL of carbonized diamine hydrochloride (Aladdin reagent Co., Ltd.) to 300. mu.L, adding 10mg/mL of N-hydroxysuccinimide (national drug group chemical Co., Ltd.) to 600. mu.L, stirring at 25 ℃ for 20min, adding 400. mu.L of 0.2mol/L of sodium carbonate solution, adding 5mL of 0.02MPBS solution containing 1.6mg/mL of goat anti-human C reactive protein antibody, stirring at 25 ℃ for 2h, adding 2mL of 2% N6-PEG (JSR Life Sciences; product number CE510) as a blocking agent, stirring for 2h, 15300rpm, centrifuging at 4 ℃ for 30min, discarding the supernatant, adding 12mL of 0.02MPBS buffer (pH 7.5) to 10min, centrifuging at 15300rpm, and resuspending at 4 ℃ for 30min, the supernatant was discarded, and 12mL of preservative solution was added for ultrasonic resuspension. Wherein the preservation solution is: contains 0.01M glycine, 0.05% 3-sulfopropylhexadecyl betaine, 1g/L sodium azide, 0.5% mannitol, and 0.5% polyethylene glycol 6000. In addition, a control reaction reagent was prepared using Bovine Serum Albumin (BSA) as a blocking agent under otherwise unchanged conditions.

Latex particles having an average diameter of 198nm were sensitized by the same preparation method as described above.

Then mixing the small-particle (68nm) sensitized emulsion reagent and the large-particle (198nm) sensitized emulsion reagent in a volume ratio of 5:1 to obtain a reaction reagent.

Example 1: method for measuring content of C-reactive protein based on immunoturbidimetry

250 μ L of the reaction reagent obtained in preparation example 1 was added to a reaction cup, incubated at 37 ℃, and then 8 μ L of CRP calibrators having different concentrations were added, respectively, and after the calibrators and the reaction reagent were mixed well, the absorbance (a1, a2) of the reaction system at a wavelength of 850nm was measured at 6 seconds and 60 seconds using an analyzer (BH-5360 CRP), respectively, and the difference (a2-a1) was calculated. And (5) taking the absorbance difference as a vertical coordinate and taking the corresponding CRP concentration as a horizontal coordinate, and making a standard curve.

The whole blood sample of the patient is measured by the same method, and the content of C-reactive protein in the sample is calculated by a standard curve.

CRP calibrants were prepared at concentrations of 175.0mg/L, 120.0mg/L, 60.0mg/L, 25.0mg/L, 5.0mg/L, and the CRP calibrants were each assayed by the method described above, three times per concentration on average, and linear regression analysis was performed on the theoretical concentration versus the average of the assayed concentrations. The results are shown in FIG. 1, where the correlation coefficient is R²When the concentration is 0.9987 and the concentration is in the range of 5-175mg/L, the theoretical value has good linear correlation with the measured value. The results show that the reaction reagent has good accuracy.

Example 2: effect of different blocking Agents on assay results

A CRP standard curve was prepared by measuring a series of concentrations of C-reactive protein standards using the reaction reagent obtained in preparation example 1 and containing N6-PEG as a blocking agent and a control reaction reagent containing Bovine Serum Albumin (BSA) as a blocking agent, respectively, by the method of example 1. As shown in Table 1 and FIG. 2, the sensitivity and upper limit of detection of the reagent were significantly improved and the linear range was wide when N6-PEG was used as the blocking agent, whereas when BSA was used as the blocking agent, the reaction intensity was only 0.6 times that of N6-PEG at a low CRP value of 5mg/mL, and the HOOK effect appeared earlier at a high CRP value and the upper limit of detection was low. Thus, it can be seen that N6-PEG is significantly superior to BSA as a blocking agent.

Table 1: test results for CRP calibrators

Example 3: effect of different hemolytic agents on the test results

Reaction reagents each containing Sodium Dodecyl Sulfate (SDS), 3-sulfopropyl-hexadecyl dimethyl betaine (Sulfobetaine-16), and hexadecyl trimethyl ammonium bromide (CTMAB) as a hemolytic agent were mixed with the CRP calibrator at a concentration of 250:8 by volume ratio, and the CRP content was measured by the method described in example 1 to prepare a CRP calibration curve. The results are shown in FIG. 3, which shows that SDS and CTMAB inhibit immunoturbidimetric reaction when used as hemolytic agent, while the reaction system using Sulfobetaine-16 as hemolytic agent has higher reactivity and wider linear range.

Example 4: detection of C-reactive protein in whole blood samples

CRP content in the whole blood sample measured using the reactive agent of preparation example 1. The whole blood sample is obtained from a fresh blood sample on the day of the hospital as anticoagulated whole blood collected with a vacuum tube. And simultaneously obtaining a serum sample of the same whole blood sample by centrifugation, and then detecting the content of the C-reactive protein in the serum sample by using a conventional C-reactive protein detection reagent (Shenzhen Meyer biomedical electronics GmbH, C-reactive protein (CRP) assay kit (latex immunoturbidimetry)) which is on the market, wherein the operation is completely carried out according to the instruction of the kit. The results are shown in FIG. 4, where the abscissa represents the CRP assay for a serum sample and the ordinate is the whole blood CRP assay for the same sample. The consistency of the two measurement results is good according to the linear regression equation, R²0.9936. The results show that the kit provided by the invention has accurate and reliable measurement results.

While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail can be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A kit, comprising:

(a) insoluble carrier particles;

(b) an antibody that specifically binds to a test substance that is a protein in a plasma fraction; and

(c) a first reagent composition comprising a sulfopropyl betaine-type surfactant and one or more reagents selected from the group consisting of: buffers, preservatives, suspending agents, stabilizers and coagulants;

wherein the kit is for immunoturbidimetric determination of the presence or amount of the substance to be tested in a blood sample and the insoluble carrier particles are provided with the antibody coated on the surface thereof in combination with the first reagent composition in suspension.

2. The kit according to claim 1, wherein the test substance is C-reactive protein (CRP).

3. The kit of claim 1, wherein the sulfopropyl betaine type surfactant is 3-sulfopropyl-hexadecyl dimethyl betaine.

4. The kit of any one of claims 1-3, which is characterized by one or more of the following features:

(i) the buffer solution is selected from glycine buffer solution, MES buffer solution, MOPSO buffer solution, Tris buffer solution, phosphate buffer solution, borate buffer solution and any combination thereof;

(ii) the preservative is selected from sodium azide, sorbate salts, benzoic acid and salts thereof, sodium nitrite and Prolin-300;

(iii) the suspending agent is selected from glycerol, ethylene glycol, mannitol, and any combination thereof;

(iv) the stabilizer is selected from sodium chloride, magnesium chloride, disodium ethylene diamine tetraacetate, bovine serum albumin, glycine, gelatin and any combination thereof;

(v) the coagulant is selected from polyethylene glycol 6000, polyethylene glycol 8000, polyethylene glycol 10000, polyethylene glycol 20000, dextran sulfate, and any combination thereof.

5. The kit of any one of claims 1-3, wherein the first reagent composition consists of: sulfopropyl betaine surfactant, antiseptic, suspending agent, coagulant, buffer solution and water.

6. The kit of any one of claims 1-3, wherein the first reagent composition consists of: 3-sulfopropylhexadecyl betaine, sodium azide, mannitol, polyethylene glycol, glycine and the balance of water.

7. The kit of any one of claims 1-3, wherein the insoluble carrier particles are latex particles.

8. The kit of claim 7, wherein the insoluble support particles are polystyrene particles.

9. A kit as claimed in any one of claims 1 to 3, wherein the insoluble carrier particles have a diameter of 50 to 400 nm.

10. The kit of any one of claims 1-3, wherein the insoluble carrier particle has a surface modified with a carboxyl, amino, hydroxyl, hydrazide, or thiol group.

11. The kit according to any one of claims 1 to 3, wherein the surface of the insoluble carrier particle is coated with an antibody that specifically binds to the substance to be tested and N6-PEG.

12. The kit of any one of claims 1-3, wherein the kit further comprises one or more reagents or devices selected from the group consisting of: a standard substance; a positive control sample; a negative control sample; an anticoagulant; and, a blood collection device.

13. An immunoturbidimetric method for determining the presence or amount of a test substance in a sample of unhemolyzed whole blood, comprising the steps of:

(1) contacting the non-hemolyzed whole blood sample with an insoluble carrier particle in a first reagent composition under conditions that allow formation of an antigen-antibody complex;

(2) measuring the turbidity change of the reaction system in the step (1);

wherein the surface of the insoluble carrier is coated with an antibody which specifically binds to the substance to be detected; the first reagent composition comprises a sulfopropyl betaine-type surfactant and one or more reagents selected from the group consisting of: buffers, preservatives, suspending agents, stabilizers and coagulants; the test substance is a protein in a plasma fraction.

14. The immunoturbidimetry of claim 13, wherein the whole blood sample is anticoagulated whole blood.

15. The immunoturbidimetry of claim 13, wherein the test agent is C-reactive protein (CRP).

16. The immunoturbidimetry of claim 13, wherein prior to step (1), further comprising the step of blocking the insoluble carrier particles with N6-PEG; alternatively, in step (1), the insoluble carrier particles are further coated with N6-PEG.

17. The immunoturbidimetry of claim 13, wherein the insoluble carrier particles are latex particles.

18. The immunoturbidimetry of claim 13, wherein the insoluble support particles are polystyrene particles.

19. The immunoturbidimetry according to claim 13, wherein in the step (2), the change in absorbance or scattered light at a predetermined wavelength within a predetermined time after the start of the reaction in the step (1) is optically measured as the change in turbidity of the reaction system in the step (1).

20. The immunoturbidimetry of claim 19, wherein the defined time is 0-5 minutes.

21. The immunoturbidimetry of claim 19, wherein the defined time is 0-2 minutes.

22. The immunoturbidimetry of claim 19, wherein the defined time is 5-120 seconds.

23. The immunoturbidimetry of claim 19, wherein the defined time period is 5-60 seconds.

24. The immunoturbidimetry of claim 19, wherein in the step (2), the absorbance of the reaction system at a predetermined wavelength is measured 2 times within 0 to 120 seconds after the start of the reaction of the step (1), and the difference is regarded as the change in turbidity.

25. The immunoturbidimetry of claim 24, wherein the absorbance of the reaction system at a prescribed wavelength is measured 2 times within 5 to 120 seconds from the start of the reaction of step (1).

26. The immunoturbidimetry of claim 24, wherein the absorbance of the reaction system at a prescribed wavelength is measured 2 times within 5 to 60 seconds from the start of the reaction of step (1).

27. The immunoturbidimetry of claim 24, wherein in the step (2), the absorbance of the reaction system at a predetermined wavelength is measured at the 6 th and 60 th seconds after the start of the reaction of the step (1), respectively, and the difference is used as the turbidity change.

28. The immunoturbidimetry of claim 19, wherein the defined wavelength is 800-900 nm.

29. The immunoturbidimetry of claim 19, wherein the defined wavelength is 840-860 nm.

30. The immunoturbidimetry of claim 19, wherein the defined wavelength is 850 nm.

31. The immunoturbidimetry of claim 13, wherein prior to step (1), further comprising one or more of the following steps: (a) obtaining a whole blood sample from a subject using a blood collection device; (b) treating the blood collection device or the whole blood sample with an anticoagulant.