CN113238060B

CN113238060B - Kit for predicting or diagnosing myocarditis

Info

Publication number: CN113238060B
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: 2022-10-11
Anticipated expiration: 2041-05-08
Also published as: CN113238060A

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 GDF15 protein or GDF15 and troponin I protein 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. The 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 degree and severity of the pathological changes, few of them are completely asymptomatic, mild ones are manifested as non-specific 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 for GDF15 are mainly focused on the assessment of the condition of heart failure and the prognosis of diseases such as myocardial infarction, cerebral infarction, etc., 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 evaluating 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 weight loss of GDF15 through a GFRAL receptor is reported in Nature Medicine by the pharmaceutical macroscopical of Li Lai, novonide and Qiangsheng. However, the use of GDF15 for the prognostic and/or diagnostic aspects of myocarditis is not known in the prior 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 GDF15 concentration level in serum or plasma for preparing a preparation for predicting or diagnosing the risk of myocarditis in an individual to be tested. When the GDF15 concentration in the serum or plasma of the subject is increased, the risk of the subject suffering from myocarditis is also increased.

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

In addition, the applicant also unexpectedly found that although the GDF15 concentration and troponin I concentration in myocarditis patients were generally higher than those of healthy people, the numerical trends of GDF15 concentration and troponin I in different myocarditis patients were inconsistent, and therefore, when GDF15 and troponin I were simultaneously detected, the accuracy of predicting or diagnosing myocarditis could 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 includes:

a GDF15 first antibody coupled to a solid support; a GDF15 secondary antibody labeled with a detectable label;

the first and second antibodies each specifically bind to a different epitope of GDF15.

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 solutions, when molecules are attacked by hydrogen peroxide ions, unstable dioxyethane is formed, which decomposes into CO2 and an electronically excited N-methylacridone, which emits photons when it 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, acridinium ester derivatives for chemiluminescence immunoassay and a method for preparing the same are described in chinese patent 201510090045.7; 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 includes: reagent 1 and reagent 2. Wherein, the reagent 1 comprises: buffer solution, inorganic salt ions, stabilizing agent, surfactant, preservative, gluconic acid and a first antibody of GDF15 coated by magnetic particles; the reagent 2 comprises: buffer, inorganic salt ions, stabilizers, surfactants, preservatives, and a second antibody to acridinium ester labeled GDF15.

Further, the kit for predicting or diagnosing myocarditis of the present invention includes: 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 GDF15 marked by acridine ester.

Further, the kit for predicting or diagnosing myocarditis of the present invention includes: 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-100.3 mL/L, 2.50mL/L of biological preservative PC-300, 11.00mL/L of glycerol and 3g/L of gluconic acid; the reagent 2 comprises: acridinium ester-labeled secondary antibody 0.025. Mu.g/L to GDF15, tris

12.11g/L, 7mL/L hydrochloric acid, 9.00g/L sodium chloride, 21.00g/L sucrose, 13.00g/L bovine serum albumin, tween-20.5 mL/L and biological preservative PC-950.00mL/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 GDF 15) and negative calibrators (not containing GDF 15).

Advantageous effects

The invention unexpectedly finds that the protein content of the GDF15 in the myocarditis patients is obviously higher than that of the common people, and the concentration trend of the GDF15 is the same as that of troponin T, so that the GDF15 can be found as a novel marker for predicting or diagnosing the 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 the advantages of 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 specifically explained below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented thereby. It will be understood by those skilled in the art that these specific embodiments and examples are illustrative of the invention and are not to be construed as limiting the invention.

The reagent components referred to in the following specific examples are commercially available products, for example, magnetic particles are available from Sichuan Michael BioNew materials technology Co., ltd. (cargo number: XCL 1026), GDF15 antibody (i.e., capture antibody for coating on magnetic particles) is available from Anti-GDF15 antibody (ab 39999) from Abcam, and GDF15 antibody (i.e., labeled antibody for acridinium ester labeling) is available from Anti-h GDF-154902SPTN-5 (100658) from Medix Biochemica.

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: and (4) CalA:0.0ng/mL; and (3) CalB:0.2ng/mL; and (4) CalC:1ng/mL; calD:5ng/mL; and (3) CalE:10ng/mL.

TABLE 1 preparation of dilution of calibrator

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 to 1L

Configuration of the kit

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

TABLE 2 formulation of the kits

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 (i 3000), 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 using the corresponding concentration value as a blank limit. The blank obtained in this example is limited to 150pg/mL.

Detection limit and quantification limit test

For the detection limit, the calibrator a to calibrator E prepared in example 1 were each measured 10 times using the kit prepared in example 1 using a full-automatic chemiluminescence immunoassay analyzer (i 3000) to calculate the average value and Standard Deviation (SD) of the measured luminescence amount at each concentration, and then 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 calibrator A to calibrator E prepared in example 1 at 0 to 10pg/mL were each measured 10 times using the kit prepared in example 1 using a full-automatic chemiluminescence immunoassay analyzer (i 3000), and the concentration at which the CV of the measurement value calculated from the standard curve is 10% or less was defined as the limit of quantitation.

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

Example 3

Linear range test

A linear sample with GDF15 concentration of about 20000pg/mL was taken and 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 300-20000pg/mL.

Example 4

Repeatability test

Enterprise-accurate reference samples (i.e., GDF15 samples at two different concentrations) were selected as assay samples. The assay was repeated 10 times for each sample 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 measurements were calculated. 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

Accuracy of enterpriseReference article	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 kit of the invention has a batch variation coefficient of less than 5%, and meets the requirement of a clinical batch variation coefficient of less than 10%.

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 invention are 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 concentrations 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 (i 3000), and the relative deviation between the values measured for the sample and the base buffer was calculated, and it was considered that there was no interference if the deviation was within 10%.

The anti-interference result of the kit of the invention is as follows: bilirubin is less than or equal to 400mg/dL; hemoglobin is less than or equal to 1250mg/dL; triglyceride is less than or equal to 3400mg/dL; biotin is less than or equal to 1000ng/dL; the rheumatoid factor is less than or equal to 1200IU/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 (i 3000), 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 myocarditis patients had significantly higher GDF15 values than 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 results of cTnI and GDF15 in different samples

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

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 are prepared from a base buffer solution containing the same GDF15 concentration, respectively, the GDF concentration in the base buffer solution not containing the above-mentioned interfering substance and in a sample prepared above is measured using a full-automatic chemiluminescence immunoassay analyzer (i 3000) using the comparison kit of the present comparative example, and the relative deviation between the measured values of the sample and the base buffer solution is calculated, and it is considered that there is no interference if the deviation is within 10%.

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

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

3. Use of a reagent for detecting the levels of GDF15 and troponin I concentrations in serum or plasma for the manufacture of a formulation for predicting or diagnosing the risk of a myocarditis in a subject to be tested.

4. A kit for predicting or diagnosing myocarditis, wherein the kit is for detecting the level of GDF15 concentration in serum or plasma; the kit comprises a reagent 1 and a 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 GDF15 marked by acridine ester.

5. The kit of claim 4, 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, 0.3mL/L of Triton X, 2.50mL/L of biological preservative PC, 11.00mL/L of glycerol and 3g/L of gluconic acid; the reagent 2 comprises: 0.025 mu g/L of secondary antibody of acridine ester labeled GDF15, 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, 200.5mL/L of Tween and 3.00mL/L of biological preservative PC-950.