阅读说明：本技术 一种通过构建桥连复合物间接检测目的分析物的检测方法 (Detection method for indirectly detecting target analyte by constructing bridging compound ) 是由 郭安亮 朱丽 赵新慧 姚见儿 于 2018-08-14 设计创作，主要内容包括：本发明提供了一种通过构建桥连复合物间接检测目的分析物的检测方法,该目的分析物可以是抗原、抗体或小分子等。通过所述方法检测目的分析物,可以有效解决化学交联方式包被标记过程中,蛋白活性位点暴露不全所致反应活性降低；部分蛋白疏水性强,化学交联方式包被标记效率低下；多种蛋白混合包被不均一,CV值大的问题；亦可保护靶向分析物,防止降解。有效提高了待测分析物的灵敏度和特异性。(The invention provides a detection method for indirectly detecting a target analyte by constructing a bridging complex, wherein the target analyte can be an antigen, an antibody or a small molecule. By detecting the target analyte by the method, the problem of reduced reactivity caused by incomplete exposure of protein active sites in the process of coating and marking in a chemical crosslinking mode can be effectively solved; partial protein has strong hydrophobicity, and the efficiency of coating and marking by a chemical crosslinking mode is low; the problems of uneven mixed coating of various proteins and large CV value; the target analyte can also be protected from degradation. Effectively improves the sensitivity and specificity of the analyte to be detected.)

1. A multiply bridged complex having the structure of formula Ia or Ib:

Z0-(A-B-C-D)n (Ia)

the elements in the formula include:

z0 is a solid phase carrier;

a is a tag protein, a protein or polypeptide fragment or a nucleotide aptamer or a small molecule and the like which are crosslinked to the surface of a solid phase carrier;

b is a first binding protein, and said B specifically binds to A;

c is a second binding protein, and said C specifically binds to B; and, one and only one of B and C is the target analyte to be detected;

d is a labeled protein, wherein D is a protein which is specifically bound with C in the formula Ia and carries a detectable label; and in formula Ib, D is a protein that specifically binds to B, and

n is more than or equal to 1;

"-" is a bond or a linking group.

2. The multiply bridged complex of claim 1, wherein the first binding protein B comprises a poorly coated protein.

3. The multiply bridged complex of claim 1, wherein the first binding protein B comprises a protein that is susceptible to degradation.

4. A detection system for detecting an analyte of interest, wherein the detection system comprises the multiply-bridged complex of claim 1.

5. The detection system of claim 4, wherein one of B and C is a detection target.

6. A kit for detecting a target analyte, the kit comprising: a container and a base reagent within the container for forming the multiply-bridged complex of claim 1, wherein the base reagent does not include a target analyte to be detected.

7. The kit of claim 6, wherein the kit comprises:

(a) a first container and ZO in said multi-component bridged composite in the first container;

(b) a second container and a of said multiply bridged complex in the second container;

(c) a third container and either B or C in said multiply bridged complex in the third container;

(d) a fourth container and D in said multiply bridged complex in the fourth container;

(e) optionally a fifth container and a buffer for the reaction system located in the fifth container;

(f) optionally a sixth container and a sample diluent in the sixth container;

(g) optionally a seventh vessel and a wash liquor located in the seventh vessel.

8. The kit of claim 7, wherein the first container, the second container, the third container, and the fourth container are the same or different containers.

9. A method for detecting a target analyte to be detected, the method comprising the steps of:

(i) providing a test system comprising a reagent starting material for forming the multiply-bridged complex of claim 1, wherein, when the test system comprises a target analyte to be detected, the reagent starting material forms the multiply-bridged complex of claim 1 with the target analyte to be detected; and

(ii) detecting the presence, absence and/or amount of said multi-component bridged complex in said detection system, thereby obtaining a detection result for said target analyte to be detected.

10. The method of claim 9, wherein the detection result comprises a qualitative and/or quantitative result.

Technical Field

The invention relates to the field of medical in-vitro diagnosis, and particularly provides a detection method for indirectly detecting a target analyte by constructing a bridging compound.

Background

The currently commonly used detection methods for antigens, antibodies and small molecules mainly comprise the following steps: 1. immunofluorescence techniques; 2. radioimmunoassay; 3. enzyme-linked immunoassay.

Immunofluorescence technology (immunofluorescence technology) is a method for qualitatively and locally examining an antigen or antibody by chemically binding a fluorescein-labeled antibody (or antigen) to a corresponding antigen (or antibody) in a tissue or a cell, and includes a direct fluorescence method and an indirect fluorescence method.

The radioimmunoassay (radioimmunoassay RIA) uses the principle of competitive binding, and should be used as a principle that a radioactive homologen labeled antigen (or antibody) is bound with a corresponding antibody (or antigen), and the method can be used for ultramicro analysis by measuring the judgment result of the radioactivity of an antigen-antibody conjugate, and can be used for measuring antigens, antibodies and antigen-antibody complexes, but the operation is unsafe and the application range is limited due to the need of using radioactive isotopes.

Enzyme-linked immunoassay (EIA) is currently the most widely used immunoassay. The specificity of antigen-antibody reaction and the high-efficiency catalytic action of enzyme on the substrate are combined, the color is developed after the enzyme acts on the substrate, the test result is judged according to the color change, the quantitative analysis can be carried out by an enzyme-labeled tester, and the sensitivity can reach ng level.

In either method, in order to obtain the relevant detection signal, there is no exception that a method of labeling an antibody or antigen with fluorescein, isotope or enzyme is used, and the capture antibody or antigen of the corresponding detection index needs to be coated or physically adsorbed on a corresponding carrier, such as a microsphere or a microplate.

Although the existing detection methods can provide detection results with certain accuracy and sensitivity for certain proteins, there is still no satisfactory detection method for certain analytes to be detected, including unstable or short-half-life analytes of interest (such as unstable proteins), and proteins whose active sites or target sites are concealed or covered during coating (including target proteins or detection reagents for detecting analytes, such as enzymes, or antigens, or antibodies, etc.).

It is known that the degradation of proteins in cells is an important process of life, most of the degradation of proteins obeys first-order reaction kinetics, the half-life period varies from tens of seconds to hundreds of days, the average turnover rate of proteins in mammalian cells is 1-2 days, the life span of key enzymes in the metabolic process and enzymes at branch points is only a few minutes, the half-life period of proteins is not constant, and the half-life period is also closely related to the physiological state of cells. Part of detection target protein is easily degraded into polypeptide fragments, which causes difficulty in subsequent detection, reduced accuracy and reduced sensitivity.

At present, the protein coating process mainly comprises the reaction of amino groups of proteins and carboxyl groups on the surface of a solid phase carrier or a microsphere. However, the protein coating method is greatly influenced by the amino acid composition of the protein, and the exposed amino content can greatly influence the coating efficiency; meanwhile, the amino sites reacting with the carboxyl groups are random, and when a large number of amino groups at the active sites of the protein are connected with the carboxyl groups of the coating carrier, the sites for specific binding of the protein are occupied and cannot be completely exposed, so that the reaction activity is greatly reduced, and the performance of a response reagent is reduced, thereby reducing the reliability and the accuracy of a detection result.

In view of the above, there is a strong need in the art to develop new, highly sensitive and highly accurate methods for detecting certain target analytes that are susceptible to degradation or are difficult to coat.

Disclosure of Invention

It is an object of the present invention to provide a method for detecting degraded or poorly coated target analytes with high sensitivity and high accuracy.

In a first aspect of the invention, a multiply bridged complex having the structure of formula Ia or Ib:

Z0-(A-B-C-D)n (Ia)

the elements in the formula include:

z0 is a solid phase carrier;

a is a tag protein, a protein or polypeptide fragment or a nucleotide aptamer or a small molecule and the like which are crosslinked to the surface of a solid phase carrier;

b is a first binding protein, and said B specifically binds to A;

c is a second binding protein, and said C specifically binds to B; and, one and only one of B and C is the target analyte to be detected;

d is a labeled protein, wherein D is a protein which is specifically bound with C in the formula Ia and carries a detectable label; and in formula Ib, D is a protein that specifically binds to B, and

n is more than or equal to 1;

"-" is a bond or a linking group.

In another preferred example, B is a target analyte to be detected.

In another preferred example, C is a target analyte to be detected.

In another preferred embodiment, the target analyte to be detected comprises an antigen, an antibody, a small molecule or a combination thereof.

In another preferred example, n is a positive integer ≧ 2.

In another preferred embodiment, n is 5 to 1X 10⁸Preferably 1X 10 to 1X 10⁷(ii) a More preferably 1X 10²-1×10⁶。

In another preferred embodiment, the solid support material is selected from the group consisting of: metal, glass, gel, plastic, or a combination thereof.

In another preferred embodiment, the solid phase carrier material comprises: a homopolymer, a copolymer, or a combination thereof.

In another preferred embodiment, the solid phase carrier material is selected from the group consisting of: polystyrene, polyethylene, polypropylene, or combinations thereof.

In another preferred embodiment, the solid phase carrier material is selected from the group consisting of: microspheres, microplates, slats, tubes, or combinations thereof.

In another preferred embodiment, the tag protein a is selected from the group consisting of: peptide fragments (including tag peptide fragments, specific polypeptide fragments (such as peptide fragments from ligands or antibodies)), aptamers, hormonal small molecules, or combinations thereof.

In another preferred embodiment, the tag protein a is selected from the group consisting of: a His tag, a GST tag, an HA tag, a c-Myc tag, a Flag tag, or a combination thereof.

In another preferred embodiment, the first binding protein B is selected from the group consisting of: an antigen, an antibody, a ligand, a receptor, or a combination thereof.

In another preferred embodiment, the first binding protein B comprises a protein that is difficult to coat.

In another preferred embodiment, the "difficult-to-coat protein" refers to a protein in which the active site or target site is masked when a protein is coated on a solid support.

In another preferred embodiment, the first binding protein B comprises a protein that is susceptible to degradation.

In another preferred embodiment, the term "readily degradable" refers to a half-life t under storage conditions of 2-8 deg.C_1/2Less than or equal to 24 hours (preferably less than or equal to 12 hours, more preferably less than or equal to 6 hours, most preferably less than or equal to 3 hours) (generally, biochemical, immunological routine test items are required to give results within 1 working day).

In another preferred embodiment, said storage conditions are such that said protein is in blood or plasma or serum and stored at 2-8 ℃.

In another preferred embodiment, the easily degradable protein comprises: gastrin Releasing Peptide (GRP).

In another preferred embodiment, the first binding protein B is located in a sample to be tested.

In another preferred embodiment, said second binding protein C is selected from the group consisting of: an antibody, an antigen, a ligand, a receptor, or a combination thereof.

In another preferred embodiment, said second binding protein C is selected from the group consisting of: IgG antibodies, IgM antibodies.

In another preferred embodiment, the marker protein D is selected from the group consisting of: an antibody, an antigen, a ligand, a receptor, or a combination thereof.

In another preferred embodiment, the labeled protein D comprises an anti-antibody.

In another preferred embodiment, the anti-antibody includes anti-human IgG antibody and anti-human IgM antibody.

In another preferred embodiment, the detectable label is selected from the group consisting of: fluorescent substances, radioactive elements, enzymes, chemiluminescent agents, colloidal gold, or combinations thereof.

In another preferred embodiment (as shown in fig. 1), in the composite,

b is a binding protein, one or more antigens that specifically bind to A;

c is protein to be detected and antibody to be detected which is specifically combined with B;

d is a labeled protein, is an anti-antibody which is specifically combined with C, and is provided with a detectable label.

In another preferred embodiment (as shown in fig. 2), in the composite,

b is a binding protein, a first antibody specifically binding to A;

c is protein to be detected and antigen to be detected which is specifically combined with B;

d is a labeled protein, a second antibody that specifically binds to C, and carries a detectable label.

In another preferred embodiment (as shown in fig. 3), in the composite,

b is a binding protein, one or more first antigens that specifically bind to A;

c is protein to be detected and antibody to be detected which is specifically combined with B;

d is a labeled protein, is a second antigen that specifically binds to C, and carries a detectable label.

In another preferred embodiment (as shown in fig. 4), in the composite,

a is a first antibody;

b is protein to be detected and antigen to be detected which is specifically combined with A;

c is a second antibody to be detected that specifically binds to B (which is used to protect antigen B);

d is a labeled protein, is an anti-antibody which is specifically combined with C, and is provided with a detectable label.

In another preferred embodiment (as shown in fig. 5), in the composite,

a is a first antibody;

b is protein to be detected and antigen to be detected which is specifically combined with A;

c is a second antibody to be detected that specifically binds to B (which is used to protect antigen B);

d is a labeled protein, is an anti-antibody which is specifically combined with B, and is provided with a detectable label.

In a second aspect of the invention, there is provided a detection system for detecting an analyte of interest, said detection system comprising a multiply-bridged complex according to the first aspect of the invention.

In another preferred embodiment, the Z0 is a microsphere (bead), a particle (particle) or a magnetic bead.

In another preferred example, in the detection system, the concentration of Z0 is 0.1 × 10 because the detection platforms are different and the deviation is large⁵To 1.0X 10⁸one/mL, preferably 1X 10⁴To 5X 10⁷one/mL, more preferably 2X 10⁴To 5X 10⁷one/mL.

In another preferred embodiment, the concentration of A in the detection system is 1-1000. mu.g/mL, preferably 5-500. mu.g/mL, more preferably 10-100. mu.g/mL.

In another preferred example, in the detection system, the concentration interval of B and/or C is 1pg/mL-1000 μ g/mL.

In another preferred embodiment, one of B and C is a detection target.

In another preferred embodiment, the concentration of D in the detection system is 0.1-1000. mu.g/mL, preferably 0.3-100. mu.g/mL, more preferably 0.5-15. mu.g/mL.

In a third aspect of the invention, there is provided a kit for detecting a target analyte, the kit comprising: a container and a starting reagent located within the container for forming a multiply-bridged complex according to the first aspect of the invention, wherein the target analyte to be detected is not included in the starting reagent.

In another preferred embodiment, the kit comprises:

(a) a first container and ZO in said multi-component bridged composite in the first container;

(b) a second container and a of said multiply bridged complex in the second container;

(c) a third container and either B or C in said multiply bridged complex in the third container;

(d) a fourth container and D in said multiply bridged complex in the fourth container;

(e) optionally a fifth container and a buffer for the reaction system located in the fifth container;

(f) optionally a sixth container and a sample diluent in the sixth container;

(g) optionally a seventh vessel and a wash liquor located in the seventh vessel.

In another preferred example, the first container, the second container, the third container and the fourth container can be the same or different containers.

In another preferred embodiment, the protein or small molecule D carries a detection label.

In another preferred embodiment, the protein or small molecule D does not carry a detection label.

In another preferred example, D in the fourth container can be not provided with a detection mark during long-term storage, and the labeling reaction is carried out within a certain time before use according to the use requirement.

In a fourth aspect of the invention, there is provided a method for detecting a target analyte to be detected, the method comprising the steps of:

(i) providing a test system comprising a starting reagent for forming the multiply-bridged complex of the first aspect of the present invention, wherein, when the test system comprises a target analyte to be detected, the starting reagent and the target analyte to be detected form the multiply-bridged complex of the first aspect of the present invention; and

(ii) detecting the presence, absence and/or amount of said multi-component bridged complex in said detection system, thereby obtaining a detection result for said target analyte to be detected.

In another preferred embodiment, the detection result comprises a qualitative and/or quantitative result.

In another preferred embodiment, the target analytes to be detected include: antigen, antibody or small molecule.

In another preferred embodiment, the target analytes to be detected include: protein, nucleic acid or small molecule compound.

In another preferred embodiment, in the detection system, the raw material reagents include: pre-complex X1 consisting of Z0, a and B.

In another preferred embodiment, in the detection system, when the target analyte to be detected is C, the raw material reagents include: (1) pre-complex X1 consisting of Z0, a and B; and (2) a D element.

In another preferred embodiment, in the detection system, the raw material reagents include: pre-complex X2 consisting of Z0 and a.

In another preferred embodiment, in the detection system, when the target analyte to be detected is B, the raw material reagents include: (1) pre-complex X2 consisting of Z0 and a; (2) c-element and (3) D-element.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows a schematic representation of a first indirectly coated or labeled bridging multiple bridging complex.

FIG. 2 shows a schematic representation of a second indirectly coated or labeled bridging multiple bridging complex.

FIG. 3 shows a schematic of a third indirectly coated or labeled bridging multiple bridging complex.

Figure 4 shows a schematic of a first protein-protected bridged multi-component bridged complex.

Figure 5 shows a schematic of a second protein-protected bridged multi-component bridged complex.

FIG. 6 is a schematic diagram showing the principle of detection in the detection of Toxoplasma gondii IgM according to the present invention.

FIG. 7 shows a schematic diagram of the detection principle in the comparative example of the toxoplasma IgM detection of the present invention.

Detailed Description

The present inventors have conducted extensive and intensive studies and extensive screening to develop for the first time a method for detecting a degraded or poorly coated target analyte with high sensitivity and high accuracy.

Firstly, the multiple bridging compound system constructed by the method enables the target protein to be indirectly combined on the solid phase carrier efficiently and specifically in the detection process, so that the target protein can be detected more sensitively. The detection system in the method can obviously improve the detection sensitivity, particularly has outstanding performance when detecting weak positive specimens, simultaneously effectively reduces the background of a reaction system, finally obviously increases the discrimination of negative and positive, can effectively reduce the probability of false positive and false negative, and has better consistency with a control kit.

Secondly, the multi-element bridging compound system constructed by the method enables the protein to be detected to be combined by the protective antibody thereof in the detection process so as to increase the stability of the protein, thereby being capable of being detected more accurately. The detection system in the method can obviously improve the sensitivity and accuracy of detection, and particularly shows that the detection system is obvious when the sample is stored for a long time.

The present invention has been completed based on this finding.

Term(s) for

As used herein, the terms "complex of the invention", "multi-bridged complex", "multi-parametric complex", or "bridged complex of the invention" and the like, are used interchangeably to refer to a complex of the first aspect of the invention having the structure shown in formula Ia or Ib. The multi-element bridging compound is particularly suitable for the situation that protein or detection reagent which is difficult to coat exists in the detection process and target analytes to be detected are easy to degrade.

Multiplexed bridged complexes

For ease of understanding, applicants provide reference to the following principles. It is to be understood, however, that the protection of the present invention is not limited by the principles described.

Taking a His antibody as an example, the basic principle of the method is that the His antibody coated on the microsphere is specifically combined with a His label connected with a C end (or an N end) of a recombinant expression antigen to obtain a microsphere-His antibody-antigen complex, after serum to be detected is added, a target antibody is captured by antigen specificity, finally a marker antibody is added to form a microsphere-His antibody-antigen-analyte-marker antibody complex (namely a multi-bridging complex), and then the complex is detected and analyzed.

Detection system

The invention also provides a detection system for detecting a target analyte, wherein the detection system comprises the multi-component bridging compound.

In another preferred embodiment, the Z0 is a microsphere (bead), a particle (particle) or a magnetic bead.

In another preferred example, in the detection system, the concentration of Z0 is 0.1 × 10 because the detection platforms are different and the deviation is large⁵To 1.0X 10⁸one/mL, preferably 1X 10⁴To 5X 10⁷one/mL, more preferably 2X 10⁴To 5X 10⁷one/mL.

In another preferred embodiment, the concentration of A in the detection system is 1-1000. mu.g/mL, preferably 5-500. mu.g/mL, more preferably 10-100. mu.g/mL.

In another preferred example, in the detection system, the concentration interval of B and/or C is 1pg/mL-1000 μ g/mL.

In another preferred embodiment, the concentration of D in the detection system is 0.1-1000. mu.g/mL, preferably 0.3-100. mu.g/mL, more preferably 0.5-15. mu.g/mL.

Detection kit

The present invention also provides a kit for detecting a target analyte, the kit comprising: a container and a raw material reagent located in the container for forming the multi-component bridging compound of the present invention, wherein the raw material reagent does not include a target analyte to be detected.

In another preferred embodiment, the kit comprises:

(a) a first container and ZO in said multi-component bridged composite in the first container;

(b) a second container and a of said multiply bridged complex in the second container;

(c) a third container and either B or C in said multiply bridged complex in the third container;

(d) a fourth container and D in said multiply bridged complex in the fourth container;

(e) optionally a fifth container and a buffer for the reaction system located in the fifth container;

(f) optionally a sixth container and a sample diluent in the sixth container;

(g) optionally a seventh vessel and a wash liquor located in the seventh vessel.

In another preferred example, the first container, the second container, the third container and the fourth container can be the same or different containers.

In the present invention, the protein or small molecule D may have a detectable label or may not have a detectable label during long-term storage, and the labeling reaction is carried out for a certain period of time before use according to the use requirement.

Detection method

The invention also provides a detection method of a target object to be detected (namely an object to be detected) based on the multi-element bridging compound.

Preferably, the analyte includes: antigens, antibodies or small molecules, etc.

In the present invention, the detection includes qualitative detection and/or quantitative detection.

In another preferred embodiment, in the detection system, the raw material reagents include: pre-complex X1 consisting of Z0, a and B.

In another preferred embodiment, in the detection system, when the target analyte to be detected is C, the raw material reagents include: (1) pre-complex X1 consisting of Z0, a and B; and (2) a D element.

In another preferred embodiment, in the detection system, the raw material reagents include: pre-complex X2 consisting of Z0 and a.

In another preferred embodiment, in the detection system, when the target analyte to be detected is B, the raw material reagents include: (1) pre-complex X2 consisting of Z0 and a; (2) c-element and (3) D-element.

The detection method can be used for scientific research, medicine research and development, medicine quality control, medicine clinical treatment monitoring, clinical patient companion diagnosis and the like.

Toxoplasma gondii detection

Toxoplasma gondii (Toxoplasma gondii) is an enteric coccidian of felines, and is found by French scholars Nicole and Manceaux in monocytes of rats right at the digits (Ctenorhodobacter gondii), and the body of the insect is Toxoplasma, and is named Toxoplasma gondii. The insect is distributed worldwide, and can infect humans and many animals, causing toxoplasmosis which is common to humans and animals, and particularly when the host immune function is low, the insect can cause serious consequences and belongs to opportunistic pathogenic protozoa (opportunistic protozoa). Toxoplasmosis includes both congenital and acquired toxoplasmosis. Congenital toxoplasmosis occurs only in the first trimester and spreads via the placental blood stream causing intrauterine infection. Most infected fetuses or babies are recessive infection, and some fetuses or babies have symptoms after birth for months or even years; can also cause abortion, premature labor, teratogenesis or stillbirth of pregnant women, especially infection in early pregnancy, and high teratogenesis rate. It has been shown that infants are symptomatic or malformed at birth with a 12% mortality rate, with 80% of survival having mental development disorders and 50% having vision disorders. Hydrocephalus, cerebral calcifications, retinochoroiditis and mental and motor disorders are typical symptoms of congenital toxoplasmosis. In addition, it is accompanied by systemic manifestations, such as fever, rash, vomiting, diarrhea, jaundice, hepatosplenomegaly, anemia, myocarditis, epilepsy, etc. in the neonatal period. Toxoplasma screening is therefore an important element of pre-pregnancy infectious disease screening. At present, enzyme-linked immunosorbent assay and chemiluminescence methods are commonly adopted in clinical diagnosis to detect specific IgG and IgM antibodies in the serum of pregnant women so as to judge the infected condition. Toxoplasma purified antigens are diagnostic antigens used earlier and are obtained by isolating and purifying tachyzoites and extracting the protein components of the cytoplasm, cell membranes and metabolites thereof. However, the purity or activity of the coated antigen is insufficient, resulting in low detection accuracy. As molecular biology techniques continue to advance, more and more genes encoding the main antigens of toxoplasma are cloned sequentially. The recombinant protein expressed by the genes has the advantages of high activity, good specificity and the like when being used as a diagnosis antigen compared with a purified antigen.

Respiratory syncytial virus detection

Respiratory Syncytial Virus (RSV) is a single-stranded negative-strand RNA virus belonging to the family Paramyxoviridae and having a diameter of about 150-300 nm. Transmitted by eye, nose and mouth secretion, and the incubation period is 2-8 days. Early infection is typically accompanied by nasal obstruction, nasal discharge, cough, wheezing, and the course of the disease can include mild rhinitis, severe respiratory depression until death. Respiratory syncytial virus is an important pathogen for respiratory infections in infants and about 80% of bronchiolitis in children and 50% of pneumonia in infants result from RSV infection and can cause Chronic Lung Disease (CLD) and asthma in children. Studies after 1970 have indicated that RSV is also a significant cause of respiratory disease in the elderly and in high-risk (immunodeficiency, lung impairment, cardiac deficiency) adult populations. Healthy elderly individuals exhibit better tolerance to RSV, while in high risk populations, admission therapy is required in 16% and a 4% mortality rate is likely to be reached.

Respiratory syncytial virus contains mainly 9 structural proteins (N, P, M, SH, G, F, M2-1, M2-2, L), of which the adhesion glycoprotein G and the fusion glycoprotein F, as two envelope proteins, mediate the fusion infection of RSV to host cells. The F protein is highly conserved in different serology and is therefore often used as a target epitope for detecting antibodies.

However, since the F protein contains a large number of hydrophobic groups, the hydrophobicity is strong, and the coating of carboxyl microspheres is not facilitated, the coating is marked in a conventional chemical crosslinking mode, and a three-parameter (microsphere-antigen-analyte-labeled antibody) detection system is low in detection signal and poor in detection sensitivity.

Hepatitis C virus detection

Hepatitis c is a disease mainly transmitted through blood, and according to the statistics of the world health organization, the global infection rate of HCV is about 3%, about 1.7 hundred million people are estimated to be infected with HCV, and about 3.5 ten thousand cases of hepatitis c are newly transmitted every year. According to statistics, about 1,000 ten thousand hepatitis C (hepatitis C) infected persons exist in China currently. Hepatitis c is a "silent" disease, with 50-90% of Hepatitis C Virus (HCV) infected individuals being asymptomatic, and over 30% of infected individuals having normal liver function. Since the initial stage of HCV infection is not readily detectable, more than 90% of infected individuals are not yet identified. Meanwhile, HCV infection has the characteristics of high concealment, high missed diagnosis and high chronicity, and 70-90% of patients can develop chronic infection. Compared with hepatitis B virus infection, chronic HCV infection is more likely to be converted into cirrhosis and even liver cancer. Therefore, HCV infection is discovered at an early stage, and the method has great significance for early diagnosis and clinical treatment of hepatitis C.

HCV belongs to flaviviridae (flaviviridae), the genome of which is single-stranded positive-strand RNA, is subject to variation, and can be currently divided into 6 genotypes and more than 50 different subtypes, and according to the international method, the HCV genotype is represented by arabic numerals, and the genotype is represented by lower case english letters (e.g., 1a, 2b, 3c, etc.). Genotype 1 is distributed globally and accounts for over 70% of all HCV infections. HCV 1b and 2a genotypes are common in China, wherein the 1b genotype is mainly used; some regions have type 1a, 2b and 3b reports; type 6 is found primarily in hong Kong and Macau, and is also found in southern border provinces.

Gastrin Releasing Peptide (GRP) assay

Gastrin Releasing Peptide (GRP) is an important regulatory factor which influences a large number of pathological and physiological processes of a human body. It is a gastrointestinal hormone, is a mammalian homologous amphibian bombesin, and was isolated from porcine gastric mucosa in 1987 and widely distributed in the nervous system, gastrointestinal tract and respiratory tract of mammals. With the dissociation of the signal peptide, its 148 amino acid preproprotein is further decomposed to produce 27 amino acid gastrin releasing peptide and 68 amino acid gastrin releasing peptide precursor (ProGRP). Due to the short half-life of GRP, about 2min, the active fraction is extremely labile in serum and therefore cannot be used in clinical assays. ProGRP is a precursor structure of GRP, is universally present in neuroendocrine cells of non-antral tissues, nerve fibers, brain and lung tissues, is a precursor with relatively stable gastrin-releasing peptide (GRP), is a novel SCLC tumor marker discovered in recent years, can be used for early discovery of SCLC, and is also helpful for judging curative effect and tumor recurrence. However, the stability of ProGRP is inferior to that of other tumor markers commonly used in clinic, such as CEA and CA 125. Studies show that the serum ProGRP has poor stability, and the degradation rate% after 24h is 30.33 +/-8.54 and the degradation rate% after 72h is 33.18 +/-8.46 after being stored at 4 ℃. After being stored at room temperature, the degradation rate is 48.59 +/-4.28 after 24 hours, and the degradation rate% is 58.41 +/-10.48 after 72 hours. The gastrin-releasing peptide precursor in serum is also degraded by endogenous proteases formed during the aggregation process, resulting in reduced sensitivity.

The main advantages of the invention include:

1) for some target analytes which are difficult to coat, the defect that the target analytes are difficult to coat and difficult to accurately detect can be overcome, and the sensitivity and the accuracy of detection are improved.

2) For some target analytes which are easy to degrade, the defect that the target analytes are difficult to accurately detect due to the easy degradation can be overcome, and the detection sensitivity and accuracy are improved.

3) And the corresponding carrier is coated by the common specific label, so that the method is convenient for industrialization, large-scale operation and reagent raw material screening, and is beneficial to controlling reagent batch difference.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

The materials and reagents used in the examples were all commercially available products unless otherwise specified.