CN113238060A

CN113238060A - Kit for predicting or diagnosing myocarditis

Info

Publication number: CN113238060A
Application number: CN202110500253.5A
Authority: CN
Inventors: 王保宁; 左旭; 刘怡; 王媛媛; 姜莱; 彭茜
Original assignee: Maccura Biotechnology Co ltd
Current assignee: Maccura Biotechnology Co ltd
Priority date: 2021-05-08
Filing date: 2021-05-08
Publication date: 2021-08-10
Anticipated expiration: 2041-05-08
Also published as: CN113238060B

Abstract

The invention relates to application of a reagent for detecting the concentration level of GDF15 in serum or plasma in preparing a preparation for predicting or diagnosing the risk of myocarditis of an individual to be detected. The invention also relates to a kit for predicting or diagnosing myocarditis, which detects the concentration of a protein that is GDF15 or GDF15 and troponin I in a subject. The invention also provides a kit for predicting or diagnosing myocarditis, which has high sensitivity, good stability, wide linear range and strong anti-interference capability and can be widely used for clinical auxiliary diagnosis of myocarditis.

Description

Kit for predicting or diagnosing myocarditis

Technical Field

The invention belongs to the technical field of clinical diagnostics, and particularly relates to a kit for predicting or diagnosing myocarditis, wherein the kit is used for determining the level of GDF15 to predict or diagnose myocarditis diseases.

Background

Myocarditis refers to a disease in which localized or diffuse inflammatory lesions of the myocardium are the main manifestation, and according to established Dallas criteria, the histological evidence of intramyocardial infiltration is myocardial inflammatory cell infiltration accompanied by adjacent myocardial cell degeneration and necrosis. Myocarditis is often caused by a common viral infection or by an immune response following a viral infection, and causes include both infectious and non-infectious groups. Infectious agents include bacteria, fungi, protozoa, parasites, spirochetes, rickettsiae and viruses. Non-infectious factors have the following aspects: (1) immune-mediated diseases (allergens, alloantigens, autoantigens); (2) toxicity (drugs, heavy metals, biologically toxic substances, physical injury, etc.). The most common cause is viral infection, with other factors being rare.

Myocarditis is clinically manifested differently, mainly depending on the wide extent and severity of the pathological changes, but few of them are completely asymptomatic, mild ones are manifested as nonspecific symptoms such as fever, cough, diarrhea, etc., and severe ones are manifested as severe arrhythmia, heart failure, cardiogenic shock and even death. Thus, the likelihood of relying solely on clinical symptoms to diagnose myocarditis is low.

For this purpose, some biological markers are used for auxiliary examination. Currently, the clinical auxiliary examination markers are mainly troponin I, troponin T or creatine kinase isozyme. However, cardiac troponin is a highly specific diagnostic marker for myocardial tissue damage (e.g., myocardial infarction), and the specificity for myocarditis remains to be studied. Therefore, more myocarditis markers are sought.

Growth differentiation factor-15 (Growth/differentiation factor 15, GDF15) is a member of the transforming Growth factor-beta superfamily (TGF beta super family). Currently, diagnostic markers of GDF15 are mainly focused on the assessment of the condition of heart failure and the prognosis of diseases such as myocardial infarction and cerebral infarction, for example, US20100167331a1 discloses the diagnosis of whether Percutaneous Cardiac Intervention (PCI) is successful in a subject with stable coronary heart disease by detecting GDF 15; US7955854B2 discloses diagnosing whether a subject exhibiting atrial fibrillation is heart failure by detecting GDF 15; predicting risk of renal failure in cardiac surgery patients by detecting GDF15 is disclosed in CN 102652261B; CN106257287B discloses a new application of growth differentiation factor 15 in the evaluation of the first stroke of hypertensive patients.

In addition, before and after 2017, the medical community also finds the potential of GDF15 in obesity treatment, and the pharmaceutical huge number of three drugs, namely Li Lai, Novonide and Qiangsheng, simultaneously report that GDF15 can lose weight through GFRAL of a receptor thereof in Nature Medicine. However, the use of GDF15 for the prediction and/or diagnosis of myocarditis is not known in the art.

Disclosure of Invention

In order to solve the above problems, the present invention provides, in a first aspect, a use of an agent for detecting a GDF15 concentration level in serum or plasma for preparing a preparation for predicting or diagnosing a risk of myocarditis in an individual to be tested. When the concentration of GDF15 in the serum or plasma of the subject is increased, the risk of myocarditis of the subject is also increased.

In another aspect of the invention, a kit for predicting or diagnosing myocarditis is provided, which is for detecting the concentration level of GDF15 in serum or plasma.

In addition, the applicant also unexpectedly found that although the concentrations of GDF15 and troponin I in myocarditis patients are generally higher than those of healthy people, the numerical trends of GDF15 and troponin I in different myocarditis patients are inconsistent, and therefore, when GDF15 and troponin I are simultaneously detected, the accuracy of predicting or diagnosing myocarditis can be improved. Namely, the kit for predicting or diagnosing myocarditis provided by the invention is used for simultaneously treating the protein concentration of GDF15 and the concentration of troponin I in serum or plasma.

Further, the kit for predicting or diagnosing myocarditis of the present invention comprises:

a GDF15 first antibody conjugated to a solid support; a GDF15 second antibody labeled with a detectable label;

the first and second antibodies each specifically bind to a different epitope of GDF 15.

In the present invention, the "solid phase carrier" may be a solid phase carrier in the form of beads (beads), microwell plates, test tubes, rods, membranes (membranes), test pieces, or the like, which are made of a material such as polystyrene, polycarbonate, polyvinyltoluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, polyacrylamide, latex, liposome, gelatin, agarose, cellulose, glass, metal, ceramic, or magnetic material. Preferably, the solid phase carriers are magnetic particles, more preferably, the solid phase carriers are magnetic particles.

In the present invention, a "detectable label" refers to a substance that can be detected spectroscopically, photochemically, biochemically, immunochemically, electrically, optically, or chemically, for example, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β -galactosidase, and other enzymes commonly used in ELISA), fluorescent dyes (e.g., fluorescein, Texas Red, rhodamine, and green fluorescent protein, etc., see, for example, Molecular Probes, Eugene, Oreg., USA), radioactive labels (e.g., 3H, 125I, 35S, 14C, or 32P), luminescent substances (e.g., chemiluminescent substances, further, such as acridine (e-9-carboxamide), phenanthridine (phenothridine), dioxetane (dioxiranes), and luminol, etc.).

Preferably, the "detectable label" is a chemiluminescent substance. "chemiluminescent substance" refers to a substance that forms an excited intermediate by catalysis by a catalyst and/or oxidation by an oxidant, and that emits photons when the excited intermediate returns to a stable ground state.

Preferably, the "detectable label" is an "acridinium chemiluminescent substance". "acridine chemiluminescent substance" refers to acridinium esters and derivatives thereof, acridinium sulfonamide chemiluminescent substances, and lucigenins, which are suitable for use in chemiluminescent immunoassay systems. In alkaline H2O2 solution, when molecules are attacked by hydrogen peroxide ions, unstable dioxyethane is generated, and the dioxyethane is decomposed into CO2 and electron excited N-methylacridone, and photons are emitted when the dioxyethane returns to the ground state.

Preferably, the "detectable label" is an "acridinium ester derivative". "acridinium ester derivatives" are well known in the field of chemiluminescence immunoassay, and for example, chinese patent 201510090045.7 describes acridinium ester derivatives for chemiluminescence immunoassay and a preparation method thereof; law et al (Novel poly-subsampled aryl acrylate esters and the use in immunoassays, J Biolumin Chemiln.1989 Jul; 4(1):88-98) synthesized a series of acridinium ester analogs that could be used in immunoassays.

Further, the kit for predicting or diagnosing myocarditis of the present invention comprises: reagent 1 and reagent 2. Wherein, the reagent 1 comprises: buffer, inorganic salt ions, stabilizers, surfactants, preservatives, gluconic acid, and a first antibody to magnetic microparticle coated GDF 15; the reagent 2 comprises: buffers, inorganic salt ions, stabilizers, surfactants, preservatives, and a second antibody to acridinium ester labeled GDF 15.

Further, the kit for predicting or diagnosing myocarditis of the present invention comprises: reagent 1 and reagent 2. Wherein, the reagent 1 comprises: 10-200 mmol/L of buffer solution, 150-600 mmol/L of inorganic salt ions, 10-50 g/L of stabilizing agent, 0.1-1.0 mL/L of surfactant, 0.1-0.6 mL/L of preservative, 2-5 g/L of gluconic acid and 0.2-0.8 ug/mL of first antibody of GDF15 coated by magnetic particles; the reagent 2 comprises: 10-200 mmol/L of buffer solution, 150-600 mmol/L of inorganic salt ions, 10-20 g/L of stabilizing agent, 0.1-1.0 mL/L of surfactant, 0.1-0.6 mL/L of preservative and 0.05-0.4 ug/L of second antibody of acridine ester labeled GDF 15.

Further, the kit for predicting or diagnosing myocarditis of the present invention comprises: reagent 1 and reagent 2. Wherein, the reagent 1 comprises: 0.5ug/mL of first antibody of GDF15 coated by magnetic particles, 2.56g/L of disodium hydrogen phosphate, 0.436g/L of sodium dihydrogen phosphate, 6.00g/L of sodium chloride, 15.00g/L of mannitol, 15.00g/L of bovine serum albumin, TX-1000.3 mL/L, biological preservative PC-3002.50 mL/L, 11.00mL/L of glycerol and 3g/L of gluconic acid; the reagent 2 comprises: second antibody 0.025. mu.g/L, Tris for acridinium ester-labeled GDF15

12.11g/L, 7mL/L hydrochloric acid, 9.00g/L sodium chloride, 21.00g/L sucrose, 13.00g/L bovine serum albumin, Tween-200.5 mL/L and biological preservative PC-9503.00 mL/L.

Further, the kit for predicting or diagnosing myocarditis of the present invention further includes a calibrator. A "calibrator," a calibration material, refers to a reference material used as an independent variable in a calibration function to provide accuracy and consistency in testing and monitoring test results in vitro diagnostic clinical tests. "calibrators" may include positive calibrators (containing GDF15) and negative calibrators (not containing GDF 15).

Advantageous effects

The invention unexpectedly finds that the protein content of GDF15 in myocarditis patients is obviously higher than that of the common people, and the concentration trend of the protein content of GDF15 is the same as that of troponin T, so that GDF15 can be found as a novel marker for predicting or diagnosing myocarditis.

The invention also provides a kit for predicting or diagnosing myocarditis, and the detection object of the kit is the protein concentration of GDF15 in serum or plasma. The kit has high sensitivity, good stability, wide linear range and strong anti-interference capability, and can be widely used for clinical auxiliary diagnosis of myocarditis.

Drawings

FIG. 1 is a graph showing GDF concentration levels in different populations (normal, myocarditis).

Detailed Description

The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.

The reagent components referred to in the following examples are commercially available products, for example, magnetic particles are available from Sichuan Meeke Biotech technologies, Inc. (Cat: XCL1026), GDF15 antibody (i.e., capture antibody for coating on magnetic particles) Anti-GDF15 antibody (ab39999) from Abcam, GDF15 antibody (i.e., labeled antibody for acridinium ester labeling) from Anti-h GDF-154902SPTN-5(100658) from Medix Biochemica, Inc.

Example 1

Preparation of calibrator

First, calibrator dilutions were prepared using the compositions and ratios shown in table 1 below, and then GDF15 antigen was added to 5 calibrator dilutions to obtain calibrators a to E, respectively. Wherein the concentrations of the antigens in the calibrator A to the calibrator E are respectively as follows: CalA: 0.0 ng/mL; and (3) CalB: 0.2 ng/mL; and (3) CalC: 1 ng/mL; CalD: 5 ng/mL; and (3) CalE: 10 ng/mL.

TABLE 1 preparation of dilutions of calibrators

Name of reagent	Dilution of calibrator
		Tris	12.11g/L
Sodium chloride	8.5g/L
		Hydrochloric acid	About 5-7mL/L
Trehalose	20.00g/L
		Bovine serum albumin	10.00g/L
TWeen-20	0.55ml/L
		Biological preservative PC-950	3.00mL/L
Purified water	Constant volume is 1L

Configuration of the kit

The kits were configured using the compositions and ratios of table 2 below.

TABLE 2 formulation of the kit

Example 2

Margin test

Using the kit prepared in example 1, the zero concentration calibrator was assayed repeatedly 20 times using a full-automatic chemiluminescence immunoassay analyzer (i3000), and the relative luminescence intensity (RLU) was recorded for 20 tests. Calculating the average value (M) and Standard Deviation (SD) of the RLU for 20 times to obtain M +2SD, substituting the value of M +2SD into the working curve of the kit (the corresponding concentration when the RLU value is M +2SD can be calculated by directly using the user software matched with the instrument), and obtaining the corresponding concentration value, namely the blank limit. The blank obtained in this example was limited to 150 pg/mL.

Detection limit and quantification limit testing

For the detection limit, the kits prepared in example 1 were used, and the calibrators a to E prepared in example 1 at 0 to 10pg/mL were each measured 10 times using a full-automatic chemiluminescence immunoassay analyzer (i3000), and after calculating the average value and Standard Deviation (SD) of the measured luminescence amount at each concentration, the lowest concentration at which the range of the measured luminescence amount average ± 3SD does not overlap the range of the luminescence amount average ± 3SD at the concentration of 0pg/mL was used as the detection limit.

For the limit of quantitation, the kits prepared in example 1 were used, and calibrators a to E prepared in example 1 at 0 to 10pg/mL were each measured 10 times using a full-automatic chemiluminescence immunoassay analyzer (i3000), and the concentration at which the CV of the measurement value calculated from the standard curve was 10% or less was defined as the limit of quantitation.

As a result, it was confirmed that GDF15 could be measured with high sensitivity, with the detection limit and the quantification limit obtained being 200pg/mL or less.

Example 3

Linear range test

A linear sample with GDF15 concentration of about 20000pg/mL was diluted with physiological saline to 9 different concentrations (dilution gradients of 0, 1/64, 1/32, 1/16, 1/8, 1/4, 1/2, 9/10, and 1, respectively) as theoretical concentrations. Each concentration was measured 3 times using the kit prepared in example 1, and the average value was calculated as the measured concentration. Calculating a linear regression equation by taking the theoretical concentration as an independent variable and the actual measurement concentration as a dependent variable, and calculating a linear regression correlation coefficient r; substituting the theoretical concentration into a linear regression equation to calculate the estimated concentration and the deviation between the measured concentration and the estimated concentration. The result shows that the linear correlation coefficient r of the kit is 1.00 and is more than 0.99; the deviation range is small, and the linear range is 300-.

Example 4

Repeatability test

An enterprise-accurate reference (i.e., two samples of GDF15 at different concentrations) was selected as the assay sample. Each sample was assayed in 10 replicates using the same batch of kits. After the measurement, the mean and standard deviation of the measurement results were calculated, respectively. And calculating the coefficient of variation of each measurement sample according to the ratio of the standard deviation to the mean value of the coefficient of variation, wherein the detection results are shown in table 3.

TABLE 3 results of repeatability tests

Enterprise precision reference product	Number of repetitions	Coefficient of variation in batch (CV)
			R1	10	3.8％
R2
		10	2.7％

As can be seen from Table 3, the kit of the invention has a variation coefficient in batch of less than 4%, and meets clinical requirements.

Intermediate precision measurement

And selecting an enterprise precision reference product as a measurement sample. Three lots of kits were taken and the assay of the samples was repeated 10 times for each lot of kits. After the measurement, the mean and standard deviation of 30 measurement results were calculated, respectively. And calculating the coefficient of variation according to the ratio of the standard deviation to the mean value of the coefficient of variation. The results are shown in Table 4.

TABLE 4 results of intermediate precision measurements

Enterprise precision reference product	Number of repetitions	Coefficient of variation between batches (CV)
			R1	30	4.2％
R2	30	3.5％

As can be seen from Table 4, the inter-batch coefficient of variation of the kit of the invention is less than 5%, and the requirement that the inter-batch coefficient of variation is less than 10% in clinic is met.

Example 5

Stability test

The calibrator with 5 concentration gradients in example 1 was placed in water baths at 4 ℃ and 37 ℃ for 0 day, 3 days, and 7 days, respectively, and then the kit of example 1 was used for detection, and the results are shown in table 5 below.

TABLE 5 stability test results

Calibration article	Signal retention rate (0 day)	Signal retention rate (3 days)	Signal retention rate (7 days)
				CalA	99％	93％	85％
CalB	98％	95％	82％
				CalC	96％	94％	83％
CalD	94％	94％	86％
				CalE	101％	98％	83％

As can be seen from Table 5, the signal retention rates of the kit of the present invention were all above 80% after being placed in a water bath at 37 ℃ for 7 days.

Example 6

Interference testing

Bilirubin solution, hemoglobin solution, triglyceride solution, biotin solution and rheumatoid factor solution of different concentration gradients were prepared from a base buffer containing the same GDF15 concentration, and the GDF concentration in the base buffer containing no interfering substance and the sample prepared above were measured using the kit of example 1 using a full-automatic chemiluminescence immunoassay analyzer (i3000), and the relative deviation between the values measured for the sample and the base buffer was calculated, and it was considered that no interference occurred within 10%.

The anti-interference result of the kit of the invention is as follows: bilirubin is less than or equal to 400 mg/dL; hemoglobin is less than or equal to 1250 mg/dL; triglyceride is less than or equal to 3400 mg/dL; biotin is less than or equal to 1000 ng/dL; the rheumatoid factor is less than or equal to 1200 IU/dL.

Example 8

Relationship of GDF15 values to myocarditis

55 myocarditis patients and 131 control groups were investigated, the control groups showing no symptoms of myocarditis and excluding the population with a known history of myocarditis disease.

Blood samples of 55 myocarditis patients and 131 control groups were tested using the kit prepared in example 1 using a full-automatic chemiluminescence immunoassay analyzer (i3000), and the GDF15 content was measured as shown in fig. 1. As can be seen from fig. 1, GDF concentration levels were not the same in different populations (normal, myocarditis patients), and it was surprisingly found that the magnitude of GDF15 was significantly higher in myocarditis patients than in the control group.

Example 9

Numerical identity of cTnI and GDF15 in patients with myocarditis

Blood samples of 40 myocarditis patients were tested using the troponin I assay kit (direct chemiluminescence method) and the kit of example 1 using a full-automatic chemiluminescence immunoassay analyzer (I3000) to obtain luminescence values as follows:

TABLE 6 detection of cTnI and GDF15 in different samples

As can be seen from the above table, although the concentrations of GDF15 and troponin I in patients with myocarditis are generally higher than those of healthy people, the numerical trends of GDF15 and troponin I in patients with different myocarditis are inconsistent, and therefore, the accuracy of predicting and/or diagnosing myocarditis can be improved by performing combined detection on cTnI and GDF 15.

Comparative example

Configuration of the contrast kit

The kits were configured using the compositions and ratios of table 7 below.

TABLE 7 formulation of comparative kits

Interference testing of contrast kits

A bilirubin solution, a hemoglobin solution, a triglyceride solution, a biotin solution and a rheumatoid factor solution having different concentration gradients were prepared from a base buffer containing the same GDF15, respectively, and the GDF concentrations in the base buffer containing no interfering substance and the sample prepared above were measured using a full-automatic chemiluminescence immunoassay analyzer (i3000) using the comparative kit of this comparative example, and the relative deviation between the values measured in the sample and the base buffer was calculated, and it was considered that no interference occurred if the deviation was within 10%.

The anti-interference result of the contrast kit is as follows: bilirubin is less than or equal to 60 mg/dL; hemoglobin is less than or equal to 1000 mg/dL; triglyceride is less than or equal to 2000 mg/dL; biotin is less than or equal to 50 ng/dL; the rheumatoid factor is less than or equal to 1200 IU/dL.

Claims

1. Use of an agent for detecting the level of GDF15 concentration in serum or plasma in the manufacture of a formulation for predicting or diagnosing the risk of myocarditis in a subject.

2. The use of claim 1, wherein the subject has an increased serum or plasma concentration of GDF15, and the subject has an increased risk of developing myocarditis.

3. A kit for predicting or diagnosing myocarditis, wherein the kit is for detecting the level of GDF15 in serum or plasma.

4. The kit according to claim 3, characterized in that it further comprises a means for detecting troponin I and/or the level of troponin I concentration in serum or plasma.

5. The kit of claim 3, wherein the kit comprises:

6. The kit of claim 3, wherein the solid support is a magnetic microparticle.

7. The kit of claim 3, wherein the detectable label is selected from the group consisting of an enzyme, biotin, digoxigenin, a colloidal metal, a fluorescent dye, and a chemiluminescent compound; preferably, the label is selected from chemiluminescent substances; preferably, the label is an acridinium ester derivative.

8. The kit of claim 3, wherein the kit comprises reagent 1 and reagent 2, wherein reagent 1 comprises: buffer, inorganic salt ions, stabilizers, surfactants, preservatives, gluconic acid, and a first antibody to magnetic microparticle coated GDF 15; the reagent 2 comprises: buffers, inorganic salt ions, stabilizers, surfactants, preservatives, and a second antibody to acridinium ester labeled GDF 15.

9. The kit according to claim 8, wherein the reagent 1 comprises: 10-200 mmol/L of buffer solution, 150-600 mmol/L of inorganic salt ions, 10-50 g/L of stabilizing agent, 0.1-1.0 mL/L of surfactant, 0.1-0.6 mL/L of preservative, 2-5 g/L of gluconic acid and 0.2-0.8 ug/mL of first antibody of GDF15 coated by magnetic particles; the reagent 2 comprises: 10-200 mmol/L of buffer solution, 150-600 mmol/L of inorganic salt ions, 10-20 g/L of stabilizing agent, 0.1-1.0 mL/L of surfactant, 0.1-0.6 mL/L of preservative and 0.05-0.4 ug/L of second antibody of acridine ester labeled GDF 15.

10. The kit of claim 3, wherein the kit comprises reagent 1 and reagent 2, wherein reagent 1 comprises: 0.5ug/mL of first antibody of GDF15 coated by magnetic particles, 2.56g/L of disodium hydrogen phosphate, 0.436g/L of sodium dihydrogen phosphate, 6.00g/L of sodium chloride, 15.00g/L of mannitol, 15.00g/L of bovine serum albumin, TX-1000.3 mL/L, biological preservative PC-3002.50 mL/L, 11.00mL/L of glycerol and 3g/L of gluconic acid; the reagent 2 comprises: 0.025 mu g/L of secondary antibody of GDF15 labeled by acridine ester, 12.11g/L of Tris, 7mL/L of hydrochloric acid, 9.00g/L of sodium chloride, 21.00g/L of sucrose, 13.00g/L of bovine serum albumin, 200.5 mL/L of Tween and 9503.00 mL/L of biological preservative PC.