CN110702929A

CN110702929A - Use of 27-hydroxycholesterol in the manufacture of a product for diagnosing schizophrenia

Info

Publication number: CN110702929A
Application number: CN201910837972.9A
Authority: CN
Inventors: 李人; 孙作厘; 王传跃
Original assignee: Beijing Anding Hospital
Current assignee: Beijing Anding Hospital
Priority date: 2019-09-05
Filing date: 2019-09-05
Publication date: 2020-01-17
Anticipated expiration: 2039-09-05
Also published as: CN110702929B

Abstract

The invention provides an application of 27-hydroxycholesterol or a reagent for detecting 27-hydroxycholesterol in preparing a reagent or a kit for diagnosing schizophrenia. The 27-hydroxycholesterol can be used as a biomarker for diagnosing schizophrenia, has higher sensitivity and specificity for diagnosing schizophrenia, can be diagnosed and predicted only by measuring one index of the peripheral blood 27-hydroxycholesterol level, and is convenient to popularize and use.

Description

Use of 27-hydroxycholesterol in the manufacture of a product for diagnosing schizophrenia

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of a brain cholesterol metabolite 27-hydroxycholesterol (27OHC) in schizophrenia diagnosis and application in preparation of a corresponding reagent.

Background

Schizophrenia is a common mental disease and is a major public health problem affecting the development of economic society. Schizophrenia has unknown etiology, poor prognosis and high disability rate, is one of the most serious and complex neuropsychiatric diseases, and has the clinical symptoms of hallucinations, delusions, neurophysiological dysfunction, neurocognitive dysfunction and the like. In recent years, development of science and technology such as neuroimaging, genomics, proteomics, immunohistochemistry and the like provides important tools for understanding the cause of schizophrenia at the gene and cell level, but the pathophysiological mechanism of schizophrenia is still unclear.

At present, most mental diseases, especially schizophrenia, lack specific pathological diagnosis indexes, clinical diagnosis can be judged by clinical experience only by depending on symptom description and duration and excluding other diseases, and diagnosis delay or misdiagnosis is very unfavorable for disease prognosis. Therefore, the search for a biomarker with high stability, specificity and sensitivity has become an important direction for the basic research and clinical research of schizophrenia.

Epidemiological results show that 60% of schizophrenic patients die from somatic diseases, and the phenomenon of increase of the incidence rate of cardiovascular diseases is particularly prominent and accounts for more than one third of the death rate of schizophrenic patients. It is reported that the risk of coronary artery disease increases 1.2 to 3.6 times and sudden cardiac death increases 6 to 8 times in patients with schizophrenia, which is 3 times higher than in the general population. The cardiovascular metabolism risk spectrum of schizophrenia patients is complex, and particularly with the wide application of atypical antipsychotics, the risk of the schizophrenia patients suffering from obesity, hyperglycemia, type II diabetes, lipid metabolism disorder and the like is obviously increased. This suggests that schizophrenia may be associated with lipid metabolism disorders in itself, and that antipsychotic therapy also causes lipid metabolism disorders, suggesting an important role for lipid metabolism disorders in schizophrenia.

Disclosure of Invention

The technical problem to be solved by the invention is that no blood biomarker is consistently recognized in schizophrenia diagnosis at present, namely, a diagnosis method for objectively judging schizophrenia is lacked in the prior art.

In order to solve the above technical problems, there is a need to screen biomarkers related to schizophrenia, thereby helping to reveal the underlying pathophysiological mechanism and developing objective diagnostic methods. There are researchers who believe that abnormal changes in total cholesterol in peripheral blood in schizophrenic patients and after treatment can be used as biomarkers, but the conclusions of different researchers are not consistent, and cholesterol cannot pass through the blood brain barrier, and the level changes and central lesions may not be directly related. It is therefore difficult to use as an objective biomarker for the diagnosis of schizophrenia.

The inventors found that peripheral cholesterol cannot freely pass through the blood-brain barrier into the center due to the presence of the blood-brain barrier, but that 27-hydroxycholesterol (27-hydroxycholesterol, 27OHC), a cholesterol metabolite, freely passes through the blood-brain barrier, and that changes in its concentration can also cause central cholesterol metabolism disorders. Thus, cholesterol metabolite 27OHC in peripheral blood has the potential to be a biomarker associated with depression.

The inventor further researches to find that the peripheral blood plasma 27OHC concentration of the schizophrenic patients is obviously reduced compared with the healthy population, and the clinical symptoms of the schizophrenic patients are obviously related to the level of the hydroxycholesterol. Plasma 27OHC levels can therefore be used as a biomarker for diagnosis in schizophrenic patients.

Based on the research findings, the invention provides the following technical scheme:

in one aspect, the present invention provides the use of 27-hydroxycholesterol or a reagent for detecting 27-hydroxycholesterol in the manufacture of a reagent or kit for the diagnosis of schizophrenia.

Preferably, the use as described above, wherein said kit is selected from the group consisting of a biochemical diagnostic kit, an immunodiagnostic kit or a molecular diagnostic kit.

Preferably, the method is used as described above, wherein the kit is one or more selected from Western blot kit, enzyme-linked immunosorbent assay (ELISA) kit, Radioimmunoassay (RIA) kit, radioimmunodiffusion kit, two-dimensional biphasic immunodiffusion kit, rocket immunoelectrophoresis kit, immunohistochemical staining kit, immunoprecipitation assay kit, complement fixation assay kit, Fluorescence Activated Cell Sorting (FACS) kit, aptamer chip kit, microarray kit and protein chip kit.

Preferably, the use as described above, wherein the kit comprises a schizophrenia rating scale; preferably, the schizophrenia rating scale is selected from one or more of the calgary schizophrenia depression scale (CDSS), positive symptoms scale, negative symptoms scale (SANS), concise negative symptoms scale (BNSS), general psychosis shape scale, and positive and negative symptoms scale (PANSS); more preferably, the schizophrenia rating scale is the positive and negative symptoms scale (PANSS).

Preferably, the above use, wherein the diagnosis of schizophrenia comprises the steps of:

step 1: determining the concentration of 27-hydroxycholesterol in the peripheral blood of the subject;

step 2: comparing the concentration of 27-hydroxycholesterol measured in step 1 with a standard concentration; and

and step 3: determining the risk of the subject suffering from schizophrenia, e.g. if the plasma 27-hydroxycholesterol concentration is below 41ng/ml, the probability of suffering from schizophrenia is about 48%;

preferably, in step 2, the standard concentration is the concentration of 27-hydroxycholesterol in peripheral blood of the normal population.

In another aspect, the present invention provides a kit for diagnosing schizophrenia, the kit comprising 27-hydroxycholesterol and a schizophrenia rating scale; preferably, the schizophrenia rating scale is selected from one or more of the calgary schizophrenia depression scale (CDSS), positive symptoms scale, negative symptoms scale (SANS), concise negative symptoms scale (BNSS), general psychosis shape scale, and positive and negative symptoms scale (PANSS); more preferably, the schizophrenia rating scale is the positive and negative symptoms scale (PANSS).

In another aspect, the present invention provides a kit for diagnosing schizophrenia, the kit comprising a reagent for detecting 27-hydroxycholesterol and a schizophrenia rating scale; preferably, the schizophrenia rating scale is selected from one or more of the calgary schizophrenia depression scale (CDSS), positive symptoms scale, negative symptoms scale (SANS), concise negative symptoms scale (BNSS), general psychosis shape scale, and positive and negative symptoms scale (PANSS); more preferably, the schizophrenia rating scale is the positive and negative symptoms scale (PANSS).

Preferably, the use or the kit, wherein the reagent for detecting 27-hydroxycholesterol is one or more selected from the group consisting of a 27-hydroxycholesterol ligand or an antibody to a 27-hydroxycholesterol ligand, an antibody to 27-hydroxycholesterol, a chromogenic reagent, a luminescent marker, a dye and a fluorescent marker.

Preferably, the use or the kit, wherein the reagent for detecting 27-hydroxycholesterol is one or more selected from the group consisting of a standard, an alkaline hydrolysis reagent, a derivatization reagent, an internal standard and an external standard; preferably, the standard is selected from serum albumin; further preferably, the alkaline hydrolysis reagent is selected from one or more of butyl hydroxy toluene, potassium hydroxide, ethylene diamine tetraacetic acid and ethanol; further preferably, the derivatization reagent is one or more than two selected from N' -diisopropyl carbodiimide, nicotinamide and 4-dimethylaminopyridine; further preferably, said internal standard is selected from D5 deuterium cholesterol and/or D7 deuterium cholesterol; further preferably, the external standard is selected from D5 deuterium cholesterol and/or D7 deuterium cholesterol.

Preferably, the use as described above or the kit as described above, wherein the kit further comprises a reagent for detecting 24-hydroxycholesterol.

The beneficial effects of the invention include:

the invention takes the level of the 27-hydroxycholesterol as the basis of the diagnosis of schizophrenia, and provides a corresponding diagnostic reagent, which has higher sensitivity and specificity for the diagnosis of schizophrenia compared with the traditional diagnostic method which can be judged by clinical experience only after depending on symptom description and duration and excluding other diseases.

On the other hand, the 27-hydroxycholesterol biomarker belongs to a peripheral blood index and can be conveniently obtained, and meanwhile, diagnosis and prediction can be carried out only by measuring one index of the peripheral blood 27-hydroxycholesterol level, so that the popularization and the use are convenient. Meanwhile, the 27-hydroxycholesterol biomarker is taken as a diagnostic method, and can be combined with other detection modes to obtain more reliable results.

Drawings

In order to more clearly illustrate the technical solution of the present invention, the drawings which are needed to be used are briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1: healthy controls, first degree relatives, clinical super high risk groups and schizophrenic patients plasma 27OHC concentrations.

FIG. 2-1: the correlation between the positive symptom score of the schizophrenic patient and the plasma 27OHC concentration is-0.301, and the concomitance probability p is 0.011.

FIG. 2-2: the correlation of negative symptom score of schizophrenia patient with plasma 27OHC concentration, r 0.064, p 0.597.

FIGS. 2 to 3: the association of the general psychotic symptom score with the plasma 27OHC concentration in schizophrenic patients, r-0.062, p-0.609.

FIGS. 2 to 4: correlation of positive and negative symptom scale scores (PANSS scores) of schizophrenic patients with plasma 27OHC concentration, r-0.051, p-0.675.

FIG. 3: 27OHC as ROC curve for diagnostic index of schizophrenia.

FIG. 4: healthy controls and schizophrenic patients plasma 24OHC concentrations.

FIG. 5-1: correlation of positive symptom score of schizophrenic patients with plasma 24OHC concentration.

FIG. 5-2: correlation of negative symptom score with plasma 24OHC concentration in schizophrenic patients.

FIGS. 5 to 3: correlation of general psychotic symptom scores with plasma 24OHC concentrations in schizophrenic patients.

FIGS. 5 to 4: correlation of schizophrenia patient positive and negative symptom scale scores (PANSS scores) with plasma 24OHC concentrations.

FIG. 6: 24OHC as a ROC curve for diagnostic markers of schizophrenia.

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In one embodiment of the invention, 27-hydroxycholesterol is used as a biomarker in a reagent or kit for diagnosing schizophrenia. Schizophrenia is diagnosed by detecting the 27OHC concentration in peripheral blood of a subject to be tested using the above-mentioned reagent or kit. Specifically, peripheral blood of a subject to be tested can be extracted, plasma can be separated, 27OHC concentration can be measured, the blood extraction time can be carried out before treatment is received so as to diagnose the diseased state of the subject, and meanwhile, the blood extraction time can also be carried out along with the treatment process or after the treatment is finished so as to monitor the treatment effect.

Herein, the term "schizophrenia" is a psychiatric disorder characterized primarily clinically by hallucinations, delusions, neurophysiological and neurocognitive dysfunction.

Herein, the term "27-hydroxycholesterol" (27-hydroxycholesterol, 27OHC) is a metabolite of cholesterol by mitochondrial cholesterol 27-hydroxylase (CYP27a1) and has the following structural formula:

27OHC is a cholesterol metabolite with the highest content in the peripheral circulation system, is finally converted into bile acid to be discharged out of the body, and plays an important role in maintaining the cholesterol balance in the body.

The term "diagnosis" refers to a method of detecting and/or identifying a pathological state in a subject, identifying and/or determining the course of whether a subject has a given disease or disorder, estimating or determining the future clinical progression of a subject, either before or after the onset of symptoms.

In a specific embodiment of the present invention, the reagent for diagnosing schizophrenia is prepared as a kit, which may be a biochemical diagnostic kit, an immunodiagnostic kit, a molecular diagnostic kit, or the like, preferably as the immunodiagnostic kit.

The diagnostic kit is prepared by adopting the principles or methods of immunology, microbiology, molecular biology and the like, and is used for diagnosing, detecting and epidemiological investigation of human diseases in vitro and the like.

The diagnostic kit may be of a type well known in the art, including, but not limited to, Western blot kit, enzyme linked immunosorbent assay (ELISA) kit, Radioimmunoassay (RIA) kit, radioimmunodiffusion, ouchterlony immunodiffusion kit, rocket immunoelectrophoresis kit, immunohistochemical staining kit, immunoprecipitation assay kit, complement fixation assay kit, fluorescence activated cell sorting (FACS kit, aptamer chip kit, microarray kit, protein chip, etc. kits.

In the kit, the reagent for detecting 27OHC is included, and the reagent refers to a molecule capable of specifically determining the concentration of 27OHC, including but not limited to nucleic acid molecules, proteins, compounds and the like that can specifically bind to 27 OHC.

In one embodiment of the present invention, a protein capable of specifically binding to 27OHC may be selected as the reagent for detecting 27OHC, and in another embodiment of the present invention, an antibody, which may be an antibody specifically binding to a marker protein, including but not limited to polyclonal antibodies, monoclonal antibodies, recombinant antibodies, and antigen-binding fragments thereof, is preferred as the reagent for detecting 27OHC, as long as it retains an antigen-binding function.

In the present invention, the reagent for detecting 27OHC may be linked with a detectable label molecule.

The present invention will be described in more detail with reference to specific examples, which, however, are for illustrative purposes only and do not limit the present invention.

The reagents and equipment sources used in the following examples are shown in Table 1 below, and reagents or equipment or procedures not described herein are routinely determined by one of ordinary skill in the art:

TABLE 1 reagents and apparatus used in the examples

Reagent/instrument	Model/specification	Manufacturer of the product
			Ammonium acetate	The content of 500g is more than or equal to 98 percent	Chemical reagent for national medicine group
Formic acid	50ml	Fluka
			Methyl tert-butyl ether	The content of 1L is more than or equal to 99.9 percent	Merck
N' -diisopropylcarbodiimide	The content of D12540725 g is more than or equal to 99 percent	Merck
			Nicotinamide	72340100 g content is more than or equal to 99 percent	Merck
4-dimethylaminopyridine	10770025 g content is more than or equal to 99 percent	Merck
			Methanol	The content of 348604L is more than or equal to 99.9 percent	Merck
High performance liquid chromatograph	Agilent G1312B	Agilent
			Mass spectrometer	AB Sciex Qtrap 6500	ABI of America

Examples

Example 1: method for collecting peripheral blood and measuring peripheral blood 27OHC concentration

1. According to the passed ethical examination experimental scheme and diagnosis standard, a healthy control population (HC), a genetic high-risk group of schizophrenia (FDR of patients), a clinical ultra-high-risk group (UHR) and a schizophrenic patient (SCZ)4 groups of 312 people are screened. The inclusion criteria for each group are as follows:

healthy control: screening of people with no mental illness or family history was performed using the American national diagnostic and statistics manual, Fourth Edition (DSM-IV) clinical Interview (Structured clinical Interview for DSM-IV Axis I Disorders-Patient Edition) and SIPS.

Schizophrenic patients were enrolled as standard: compliance with the American handbook of diagnosis and statistics for mental disorders, four edition diagnostic criteria (DSM-IV), history of other mental disorders other than schizophrenia, neurological and other severe physical disorders without definitive diagnosis; there was no family history of neurological disease. Eliminating extreme excitement, impulsion and non-matching authors; patients who have been treated by other scientific research or MECT within three months. The first-degree relatives of the patients exclude neurological and mental diseases and other serious somatic diseases from being grouped into a genetic high-risk group.

Clinical high risk mental illness risk syndrome institutional interview (SIPS) screening one or more of the three conditions that meet mental illness risk syndrome criteria.

All subjects were 14-55 years old, excluding pregnant or lactating women, those with food drug allergies, those who could not discontinue use of drugs due to other somatic diseases, history of alcohol and drug abuse, and history of head trauma.

Patients and normal subjects were screened using a DSM-IV Clinical Interview (SCID), and patients were assessed for Clinical psychopathological symptoms using a positive and negative scale (PANSS). Simultaneously, 5ml of peripheral blood is extracted into an EDTA anticoagulant tube, centrifuged at 3000 rpm/min for 10 min, and upper plasma is separated, immediately subpackaged and stored in an ultra-low temperature refrigerator at minus 80 ℃ for later use.

2. Extracting blood plasma, and detecting 27OHC content by high performance liquid chromatography-mass spectrometry, wherein the specific method comprises the following steps:

(1) a standard solution with 27OHC concentration of 1-2000ng/ml was prepared from 5% bovine serum albumin solution, and 5% bovine serum albumin solution was used as a blank sample.

(2) To a 1.5ml EP tube was added 50. mu.l standard/blank/plasma to be tested, followed by 50. mu.l internal standard (D5 deuterium cholesterol + D7 deuterium cholesterol 50ng/ml), followed by 200. mu.l acidic buffer (50mM ammonium acetate, 1% formic acid) and 1ml methyl tert-butyl ether.

(3) After mixing well, the sample was placed in a-80 ℃ freezer, and after the aqueous phase was cooled and crystallized, 0.5ml of methyl tert-butyl ether phase (supernatant) was transferred to a 1.5ml centrifuge tube.

(4) Methyl tert-butyl ether was blown dry at 35 ℃ under nitrogen.

(5) To each EP tube was added 50. mu.l of a derivatizing agent (63mg of N, N' -diisopropylcarbodiimide, 62mg of nicotinamide and 61mg of 4-dimethylaminopyridine in 5ml of chloroform), and mixed by heating at 35 ℃ for 2 hours.

(6) The dichloromethane was blown dry with nitrogen at 35 ℃.

(7) Sample detection was performed after 100. mu.l methanol reconstitution.

In the following examples, peripheral blood was collected and the concentration of 27OHC in peripheral blood was measured by the method described in the present example.

Example 2: peripheral blood 24OHC or 27OHC as biomarker for schizophrenia diagnosis

1. The study included a total of 312 gender-matched subjects, and the basic demographics and patient symptom assessments for each group are shown in table 2.

Table 2: assessment of basic population and clinical symptoms in patients

Note: HC: a healthy control; FDR: first degree relatives of schizophrenia; UHR: the clinical ultra-high risk of schizophrenia; SCZ: patients with schizophrenia; and (4) SIPs: a psychiatric risk syndrome institutional interview; PANSS: positive and negative symptom scales; p: positive symptoms; n: negative symptoms; g: general psychotic symptoms: t: total points; NA: missing values.

By detecting the plasma 27OHC, the plasma 27OHC level of the schizophrenic patients is obviously reduced compared with that of healthy control groups (figure 1, p <0.0001), which indicates that the cholesterol metabolism of the patients is obviously reduced and can be a potential biomarker for the diagnosis of the schizophrenic diseases. However, there were no significant changes in the first-degree relatives and the clinically very high-risk group of schizophrenia compared to the healthy control group (fig. 1), indicating that the reduction in schizophrenia by 27OHC did not occur before onset. The results of the correlation analysis showed that clinical symptoms of schizophrenic patients were significantly correlated with 27OHC levels (fig. 2-1 to fig. 2-4), wherein 27OHC was negatively correlated with positive symptoms (fig. 2-1), further demonstrating the relationship between the changes in 27OHC levels and the development of the disease. A Receiver Operating Characteristic curve (ROC) curve (figure 3) was prepared by combining the family history index of the subject and 27OHC as diagnostic indexes, the area under the curve was 0.741, and the diagnostic sensitivity and specificity were 48.0% and 88.9%, respectively.

Through plasma 24OHC detection, the plasma 24OHC of the schizophrenic patients is obviously increased compared with that of healthy control groups (figure 4, p <0.05), which indicates that the cholesterol metabolism in the brains of the patients is obviously increased and can be a potential biomarker for schizophrenic diagnosis. The correlation analysis result shows that the clinical symptoms of the schizophrenic patients are obviously correlated with the hydroxycholesterol level (fig. 5-1 to fig. 5-4), wherein, 24OHC is positively correlated with the negative symptoms and the total PANSS component, and further proves the relationship between the change of the hydroxycholesterol level and the occurrence and development of the diseases. The Receiver Operating Characteristic curve (ROC) curve (FIG. 6) was prepared by combining the family history index of the subject and the concentration of 24OHC as the diagnostic index, the area under the curve was 0.703, and the diagnostic sensitivity and specificity were 71.6% and 61.1%, respectively.

As can be seen from the above examples, the present invention diagnoses the condition of schizophrenia patients by selecting peripheral blood 27OHC or 24OHC as a biomarker, realizes an objective diagnosis method for schizophrenia, and has high sensitivity and specificity for schizophrenia diagnosis.

Claims

Use of 27-hydroxycholesterol or a reagent for detecting 27-hydroxycholesterol in the manufacture of a reagent or kit for the diagnosis of schizophrenia.
2. The use according to claim 1, wherein the kit is selected from a biochemical diagnostic kit, an immunodiagnostic kit or a molecular diagnostic kit.
3. The use of claim 1 or 2, wherein the kit is selected from one or more of a Western blot kit, an enzyme-linked immunosorbent assay (ELISA) kit, a Radioimmunoassay (RIA) kit, a radioimmunodiffusion kit, a two-dimensional biphasic immunodiffusion kit, a rocket immunoelectrophoresis kit, an immunohistochemical staining kit, an immunoprecipitation assay kit, a complement fixation assay kit, a Fluorescence Activated Cell Sorting (FACS) kit, an aptamer chip kit, a microarray kit, and a protein chip kit.
4. The use according to any one of claims 1-3, wherein the kit comprises a schizophrenia rating scale; preferably, the schizophrenia rating scale is selected from one or more of the calgary schizophrenia depression scale (CDSS), positive symptoms scale, negative symptoms scale (SANS), concise negative symptoms scale (BNSS), general psychosis shape scale, and positive and negative symptoms scale (PANSS); more preferably, the schizophrenia rating scale is the positive and negative symptoms scale (PANSS).
5. The use according to claim 1, wherein the diagnosis of schizophrenia comprises the steps of:

step 1: determining the concentration of 27-hydroxycholesterol in the peripheral blood of the subject;

step 2: comparing the concentration of 27-hydroxycholesterol measured in step 1 with a standard concentration; and

and step 3: determining the risk of the subject suffering from schizophrenia, e.g. if the plasma 27-hydroxycholesterol concentration is below 41ng/ml, the probability of suffering from schizophrenia is about 48%;

preferably, in step 2, the standard concentration is the concentration of 27-hydroxycholesterol in peripheral blood of the normal population.
6. A kit for diagnosing schizophrenia, the kit comprising 27-hydroxycholesterol and a schizophrenia rating scale; preferably, the schizophrenia rating scale is selected from one or more of the calgary schizophrenia depression scale (CDSS), positive symptoms scale, negative symptoms scale (SANS), concise negative symptoms scale (BNSS), general psychosis shape scale, and positive and negative symptoms scale (PANSS); more preferably, the schizophrenia rating scale is the positive and negative symptoms scale (PANSS).
7. A kit for diagnosing schizophrenia, the kit comprising reagents for detecting 27-hydroxycholesterol and a schizophrenia evaluation scale; preferably, the schizophrenia rating scale is selected from one or more of the calgary schizophrenia depression scale (CDSS), positive symptoms scale, negative symptoms scale (SANS), concise negative symptoms scale (BNSS), general psychosis shape scale, and positive and negative symptoms scale (PANSS); more preferably, the schizophrenia rating scale is the positive and negative symptoms scale (PANSS).
8. The use according to any one of claims 1 to 5 or the kit according to claim 7, wherein the reagent for detecting 27-hydroxycholesterol is selected from one or more of 27-hydroxycholesterol ligand or an antibody to 27-hydroxycholesterol ligand, an antibody to 27-hydroxycholesterol, a chromogenic reagent, a luminescent marker, a dye and a fluorescent marker.
9. The use according to any one of claims 1 to 5 or the kit according to claim 7, wherein the reagent for detecting 27-hydroxycholesterol is selected from one or more of a standard, an alkaline hydrolysis reagent, a derivatizing reagent, an internal standard and an external standard; preferably, the standard is selected from serum albumin; further preferably, the alkaline hydrolysis reagent is selected from one or more of butyl hydroxy toluene, potassium hydroxide, ethylene diamine tetraacetic acid and ethanol; further preferably, the derivatization reagent is one or more than two selected from N' -diisopropyl carbodiimide, nicotinamide and 4-dimethylaminopyridine; further preferably, said internal standard is selected from D5 deuterium cholesterol and/or D7 deuterium cholesterol; further preferably, the external standard is selected from D5 deuterium cholesterol and/or D7 deuterium cholesterol.
10. The use according to any one of claims 1 to 5 or the kit according to any one of claims 6 to 9, wherein the kit further comprises reagents for detecting 24-hydroxycholesterol.