Kit and method for evaluating and / or predicting skeletal development condition

Abstract

The invention provides a kit and a method for evaluating and / or predicting skeletal development conditions. The kit comprises a reagent for detecting the sulfate content, and the sulfate content serves as a biomarker of the skeletal development condition. The sulfate content is used as the biomarker for evaluating and / or predicting the skeletal development condition, and the biomarker has the advantages of high sensitivity, high specificity, low detection cost, convenience in material taking, easiness in sample storage and the like, can be widely applied to related work such as health examination and disease general survey, and becomes an effective means for growth and development evaluation and auxiliary disease diagnosis. Moreover, the biomarker disclosed by the invention has the characteristics that the smaller the age (before adolescence) is, the higher the correlation is, so that the higher the sensitivity is, which means that the biomarker disclosed by the invention can be used for effectively evaluating and / or predicting the skeletal development condition, and identifying and intervening diseases earlier, and is more likely to improve the final height of a patient with short stature.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical detection and specifically relates to a kit and method for evaluating and / or predicting bone development. Background Art

[0002] Growth monitoring and evaluation of infants and children is part of the child health program. Linear growth during childhood is affected by many factors (e.g., prenatal, nutritional, hormonal, environmental or genetic). Problems in any aspect of skeletal development will affect normal growth and development, resulting in short stature and growth retardation, which is a relatively early sign of poor health. Short stature (SS) is usually defined as a height that is more than 2 standard deviations (SDS) below the mean for the same age and sex. In addition to short stature caused by malnutrition, which depends on the socioeconomic environment, primary or secondary short stature without obvious manifestations has a certain prevalence in different countries. The cause of 60-80% of children with short stature cannot be determined and is classified as idiopathic short stature. These children are born with normal height, weight and body proportions, and no evidence of systemic, endocrine, nutritional, chromosomal abnormalities or genetic mutations is found.

[0003] At present, there are still great challenges in the diagnosis of short stature. On the one hand, they usually show similar phenotypes, and on the other hand, there is still a lack of comprehensive understanding of the pathogenic factors. Therefore, only a small number of patients with affected stature receive appropriate treatment each year. In addition, the final result of bone growth is irreversible. With the increase of age, the treatment of short stature becomes more difficult and the space for further bone growth becomes more limited. Based on the evaluation and / or prediction of bone development status, understanding the genetic causes of growth disorders can directly enhance the molecular understanding of the mechanism of bone growth in children, and continuously expand the molecular level diagnosis ability of clinicians for SS in children with unknown etiology, so as to obtain more precise treatment guidelines and avoid expensive diagnosis and treatment costs.

[0004] The means of evaluating and / or predicting the status of bone development is an important part of the growth monitoring of infants and children and the early identification of skeletal dysplasia and short stature to improve the treatment process. The evaluation and / or prediction results of skeletal development status can obtain effective growth and development information, assisting clinicians to further select standard methods and means to obtain the final disease screening, diagnosis, characterization and monitoring conclusions. Biomarkers have been widely used in biological observations. As intermediate results obtained from healthy people and patients that are usually difficult to observe by other detection methods, they can provide effective guidance for subsequent disease diagnosis and treatment and growth and development monitoring. Therefore, finding effective biomarkers for evaluating and / or predicting skeletal development status has always been an urgent problem to be solved in the field of biomedical testing. Summary of the invention

[0005] In view of the above problems, the inventors have found through research that there is a strong correlation between the sulfate content in serum or plasma and height, and this correlation is applicable to both healthy groups and all bone development disorders, especially patients with short stature, and the younger the target group, the higher the correlation. Therefore, the present invention provides a kit for evaluating and / or predicting bone development status, which includes a reagent for detecting sulfate content, wherein sulfate content is used as a biomarker for bone development status.

[0006] The above object of the present invention is achieved by providing the following technical solutions:

[0007] In a first aspect, the present invention provides a kit for evaluating and / or predicting bone development status, which comprises a reagent for detecting sulfate content, wherein the sulfate content serves as a biomarker for bone development status.

[0008] According to some embodiments of the invention, the skeletal development condition is a mammalian skeletal development condition.

[0009] Preferably, the mammal is selected from one or more of humans, monkeys, mice and pigs, preferably humans, and more preferably infants and / or children.

[0010] According to some embodiments of the present invention, the evaluation and / or prediction of skeletal development status is the diagnosis and / or early prediction of skeletal development disorders and related diseases.

[0011] According to some embodiments of the present invention, the kit is a kit for detecting sulfate content by spectrophotometry.

[0012] According to some embodiments of the invention, the kit further comprises means for obtaining serum and / or plasma samples.

[0013] According to some embodiments of the invention, the biomarker is sulfate content in serum and / or plasma.

[0014] According to some embodiments of the present invention, the reagents include a standard sulfate aqueous solution of known concentration, anhydrous ethanol, and a working reagent.

[0015] Preferably, the concentration of the standard sulfate aqueous solution is 0.8-1.1 mg / ml.

[0016] Preferably, the working reagent comprises a barium salt and polyethylene glycol.

[0017] More preferably, the barium salt is barium chloride.

[0018] More preferably, the polyethylene glycol is polyethylene glycol 4000.

[0019] Further preferably, the molar ratio of the barium salt to the polyethylene glycol in the working reagent is 0.8-1:1.

[0020] In a second aspect, the present invention provides use of a reagent for detecting sulfate content in the preparation of a kit for evaluating and / or predicting bone development status, wherein the sulfate content serves as a biomarker for bone development status.

[0021] According to some embodiments of the invention, the skeletal development condition is a mammalian skeletal development condition.

[0022] Preferably, the mammal is selected from one or more of humans, monkeys, mice and pigs, preferably humans, and more preferably infants and / or children.

[0023] According to some embodiments of the present invention, the evaluation and / or prediction of skeletal development status is the diagnosis and / or early prediction of skeletal development disorders and related diseases.

[0024] According to some embodiments of the present invention, the kit is a kit for detecting sulfate content by spectrophotometry.

[0025] According to some embodiments of the invention, the kit further comprises means for obtaining serum and / or plasma samples.

[0026] According to some embodiments of the invention, the biomarker is sulfate content in serum and / or plasma.

[0027] According to some embodiments of the present invention, the reagents include a standard sulfate aqueous solution of known concentration, anhydrous ethanol, and a working reagent.

[0028] Preferably, the concentration of the standard sulfate aqueous solution is 0.8-1.1 mg / ml.

[0029] Preferably, the working reagent comprises a barium salt and polyethylene glycol.

[0030] More preferably, the barium salt is barium chloride.

[0031] More preferably, the polyethylene glycol is polyethylene glycol 4000.

[0032] Further preferably, the molar ratio of the barium salt to the polyethylene glycol in the working reagent is 0.8-1:1.

[0033] According to some embodiments of the present invention, the skeletal development disorder and related diseases thereof are selected from one or more of short stature, constitutional growth retardation, osteochondrodysplasia and psychosocial dwarfism, preferably short stature.

[0034] Preferably, the short stature is selected from one or more of familial short stature, idiopathic short stature, short stature due to chromosomal abnormalities, short stature due to Prader-Willi syndrome, short stature due to Russell-Silver syndrome, short stature due to Noonan syndrome, short stature due to chronic systemic diseases, short stature due to growth hormone deficiency, short stature due to hypothyroidism, short stature due to precocious puberty and short stature due to Cushing's syndrome.

[0035] Preferably, the short stature is short stature in infants, children and / or adolescents.

[0036] In a third aspect, the present invention provides a method for evaluating and / or predicting skeletal development for non-diagnostic purposes, comprising the following steps:

[0037] (1) Determine the sulfate content in samples from subjects and healthy controls, and record them as measured values ​​and reference values;

[0038] (2) Using the sulfate content as a biomarker for bone development status, and comparing the measured value with a reference value to evaluate and / or predict the bone development status.

[0039] According to some embodiments of the invention, the skeletal development condition is a mammalian skeletal development condition.

[0040] Preferably, the mammal is selected from one or more of humans, monkeys, mice and pigs, preferably humans, and more preferably infants and / or children.

[0041] According to some embodiments of the invention, the method detects sulfate content by spectrophotometry.

[0042] According to some embodiments of the invention, the sample is serum and / or plasma.

[0043] Preferably, the method is carried out using a kit according to the first aspect of the present invention.

[0044] The present invention has at least the following beneficial effects:

[0045] The present invention discovered for the first time that there is a strong correlation between the sulfate content in serum or plasma and height, and this correlation is applicable to both healthy groups and all bone development disorders, especially patients with short stature. Moreover, the biomarker of the present invention has the characteristics that the younger the age (prepubertal), the higher the correlation, and thus the higher the sensitivity, which is of great significance for patients with bone development disorders, especially short stature, and means that the use of the biomarker of the present invention can effectively evaluate and / or predict the state of bone development, as well as identify and intervene in the disease earlier, and is more likely to improve the final height of patients with short stature.

[0046] The present invention selects the sulfate content in serum / plasma as a biomarker of bone development, which shows a strong correlation with height in both human samples (n=948) and pig samples (n=74). This biomarker has the advantages of high sensitivity, strong specificity, low detection cost, convenient material collection, and easy sample storage (serum / plasma can be stored at -20°C). It can be widely used in health examinations and disease surveys and other related work, and has become an effective means of growth and development evaluation and auxiliary disease diagnosis.

[0047] In addition, the sulfate content in serum / plasma as a new biomarker of bone development status will overcome the problems that the current diagnostic methods for this type of disease cannot achieve early diagnosis or have low diagnostic specificity and sensitivity. It can significantly improve the clinical detection rate of the disease by existing methods and achieve early diagnosis and treatment of the disease and effective monitoring of growth and development.

[0048] In addition, the biomarkers of the present invention have high specificity in different ages, genders, and developmental stages, which means that the biomarkers are more applicable. And the use of the biomarkers of the present invention greatly reduces the pain and cost of the traditional detection methods for healthy groups and patients, and to a certain extent provides material convenience for health monitoring and disease treatment and prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0050] Figure 1 It showed that the sulfate content in the plasma of patients with short stature was significantly lower than that of the normal control group;

[0051] Figure 2 Shows the abnormalities of bone development in patients with short stature; R stands for right; L stands for left;

[0052] Figure 3 It shows that height and sulfate content in the population sample are strongly correlated; among them, Pearson represents the correlation coefficient; CI 95%represents the 95% confidence interval;

[0053] Figure 4 It shows that there is a strong correlation between height and sulfate content in male samples; among them, Pearson represents the correlation coefficient; CI 95% represents the 95% confidence interval;

[0054] Figure 5 It shows that height and sulfate content in female samples are strongly correlated; among them, Pearson represents the correlation coefficient; CI 95% represents the 95% confidence interval;

[0055] Figure 6 Logical analysis showing the correlation between different influencing factors in the pig sample; where FCR stands for feed conversion ratio, ADFI stands for average daily feed intake, ADG stands for average daily gain, and FCR is the ratio of ADFI to ADG;

[0056] Figure 7 The height of the pig samples was strongly correlated with the sulfate content; Pearson represents the correlation coefficient; CI 95% represents the 95% confidence interval. DETAILED DESCRIPTION

[0057] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention.

[0058] Example 1 Detection of sulfate content in serum or plasma

[0059] 1. Reagent Preparation

[0060] a) Sulfate standard solution: Weigh 1.4786 g of anhydrous sodium sulfate (Na2SO4, premium grade) or 1.81411 g of anhydrous potassium sulfate (K2SO4, premium grade), dissolve in a small amount of water, place in a 1000 ml volumetric flask, and dilute to the mark. The standard solution contains 1 mg / ml, i.e. 10 mM sulfate ions (SO4 2- ).

[0061] b) Working Reagent (WR): WR should be used within 1 hour after preparation. The preparation method is as follows: 1.8 ml of barium chloride (0.1 mol / L) is mixed with 2 ml of polyethylene glycol 4000 (PEG4000, 0.1 mol / L), and then 16.2 ml of ddH2O is added and mixed thoroughly to ensure that the powder is completely dissolved. Prepare enough WR for all samples and standards, that is, 100 μL of WR per well, and equilibrate the freshly prepared working reagent for 10 minutes before use.

[0062] 2. Experimental Procedure

[0063] a) Sample preparation: Fresh serum or plasma (non-hemolyzed) samples can be tested immediately or frozen for future testing.

[0064] b) Deproteinization of samples: 200 μL of sample was mixed with 100 μL of anhydrous ethanol solution in a 1.5 ml centrifuge tube, and the protein was precipitated at 14,000 rpm in a low-temperature tabletop centrifuge for 5 min, and then 200 μL of the supernatant was transferred for analysis.

[0065] c) Prepare the standard: Mix 100 μL of 10 mM sulfate standard solution with 400 μL of ddH2O to prepare a 2.0 mM premix. Dilute the premix in a gradient as shown in the table below.

[0066] Table 1 Dilution gradient of premix

[0067]

[0068] d) Transfer 200 μL of the standard to different wells of a transparent 96-well plate. Note: When 100 μL of anhydrous ethanol is added to the sample for deproteinization, add 100 μL of anhydrous ethanol to each 200 μL standard, mix and transfer 200 μL of the resulting standard to different wells.

[0069] e) Transfer 200 μL of sample (i.e., deproteinized supernatant) to individual wells of a 96-well plate.

[0070] f) Using a multichannel pipette, add 100 μL of working reagent to each well and vortex gently to mix. If anhydrous ethanol was added to the sample or standard in the above step, mix the sample or standard with WR by pipetting up and down once to improve.

[0071] g) Incubate at room temperature for 5 min and read the optical density at 540–610 nm (preferably 600 nm).

[0072] 3. Calculation method of sulfate content (mM) in samples

[0073] The concentration of a series of sulfate standards is used as the horizontal axis and the optical density value is used as the vertical axis. The fitting straight line equation, i.e., the standard curve, is obtained through linear regression analysis.

[0074] Sulfate content = (OD 样品 –OD ddH2O ) / x;

[0075] OD 样品 and OD ddH2O OD values ​​of the sample well and ddH2O well, respectively;

[0076] x is the slope of the standard curve obtained using the standard.

[0077] Example 2 Correlation Analysis Method

[0078] 1. Sample source

[0079] (1) Population sample

[0080] Among the 955 children, 2 children without physical growth records, 3 children with height standard deviation score (HtSDS) <-4 SD, and 2 children with body mass index (BMI) standard deviation score (BMISDS) >4 SD were excluded, and the remaining 948 children were included in the data analysis. The average age of the 948 children was 13.5±2.2 years (age span was 8.94-19.00 years), including 429 boys (45.3%) and 519 girls (54.7%). Among these children, there were 129 children with short stature, including 54 boys and 75 girls. After measurement, the height range of these children was 110.6-189.6 cm, which was normally distributed. The plasma sulfate content of these children measured according to the method of Example 1 ranged from 0.04-0.65 mM, which was normally distributed.

[0081] In addition, according to the development of female breasts, the female samples were divided into 6 stages (Tanner development stage standard), namely 0, B1, B2, B3, B4 and B5. According to the development of male testicles, the male samples were divided into 6 stages (Tanner development stage standard), namely 0, I, II, III, IV and V.

[0082] Based on the above information, the present invention selects the following indicators for analyzing population samples: gender, age, sulfate content, height, weight, body mass index (BMI), waist circumference and hip circumference, and uses the stepwise logistic regression method to analyze which factor is significantly correlated with sulfate.

[0083] (2) Pig herd samples

[0084] The present invention collected 74 pigs, including 43 boars and 31 sows, with an age of about 180 days. The height of the 74 pigs was measured to be 58-67 cm, showing a normal distribution. The plasma sulfate content of the 74 pigs measured by the method of Example 1 was in the range of 0.88-1.38 mM.

[0085] Based on the above information, the present invention selects the following indicators for analyzing the pig samples: height, body length, weight, back fat, eye muscle, sulfate content, FCR, ADFI and ADG.

[0086] 2. Stepwise Logistic Regression Method

[0087] For the above data, the stepwise logistic regression method is used to analyze which factor is significantly correlated with sulfate content. Specifically, a stepwise regression analysis based on AIC (Akaike Information Criterion) of the Backward Stepwise Selection method is introduced. The independent variables are introduced one by one, and the condition for the introduction is that their partial regression sum of squares is significant after testing. At the same time, after each new independent variable is introduced, the old independent variables are tested one by one, and the independent variables with insignificant partial regression sum of squares are eliminated. In this way, new variables are introduced and eliminated at the same time until no new variables are introduced and no old variables are deleted. Each variable is analyzed step by step, and each time the variable that can minimize the AIC value is selected to be added to the model until the addition of any variables not included does not result in a significant decrease in the AIC value.

[0088] 3. Analyze the results

[0089] The analysis results are shown in Tables 2-4 and Figure 1-7 shown.

[0090] Table 2 Correlation between height, gender, age and plasma sulfate content in population samples

[0091]

[0092] The above table shows that age and height in the population sample are strongly correlated with the sulfate content in plasma.

[0093] Table 3 Correlation between relevant indicators at different developmental stages and plasma sulfate content in male samples

[0094]

[0095] It can be seen from the above table that height, age and plasma sulfate content in male samples are strongly correlated.

[0096] Table 4 Correlation between relevant indicators at different developmental stages and plasma sulfate content in female samples

[0097]

[0098] It can be seen from the above table that height, age and plasma sulfate content in female samples are strongly correlated.

[0099] Depend on Figure 1 It can be seen that the sulfate content in the plasma of patients with short stature is significantly lower than that of the normal control group.

[0100] Depend on Figure 2 It can be seen that patients with short stature have abnormal bone development.

[0101] Depend on Figure 3 It can be seen that there is a strong correlation between height and sulfate content in the population sample.

[0102] Depend on Figure 4 It can be seen that the height and plasma sulfate content in male samples are strongly correlated, among which the correlation coefficients of stages 0 to III are 0.73, 0.74, 0.80 and 0.75, respectively, which are all higher than the correlation coefficients of stages IV and V. It can be seen that the plasma sulfate content in male samples has the characteristics of higher correlation and higher sensitivity as the age is younger (before puberty).

[0103] Depend on Figure 5 It can be seen that height and plasma sulfate content in female samples are strongly correlated, among which the correlation coefficients of stage 0 and B1 are 0.85 and 0.88 respectively, which are higher than the correlation coefficients of stages B2 to B5. It can be seen that the plasma sulfate content in female samples has the characteristics of higher correlation and higher sensitivity as the age is younger (before puberty).

[0104] Depend on Figure 6 It can be seen that in the correlation analysis of the nine indicators of the pig samples (namely weight, height, sulfate content, body length, ADFI, FCR, ADG, back fat and eye muscle), height has the highest correlation with sulfate content, and the height of pigs corresponding to samples with lower sulfate content is also relatively low.

[0105] Depend on Figure 7 It can be seen that there is a strong correlation between height and sulfate content in pig samples, with a correlation coefficient of 0.77.

[0106] In summary, through the above correlation analysis, it was found that there is a strong correlation between the sulfate content in serum or plasma and height, which can be used as a biomarker for bone development.

[0107] The above are only several exemplary embodiments of the present invention, and are not intended to limit the present invention in any form. Although the present invention is disclosed as above with preferred embodiments, they are not intended to limit the present invention. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, can make some changes or modifications to obtain equivalent or equivalent embodiments using the above disclosed technical content, all of which belong to the scope of the present invention.

Claims

1. A kit for evaluating and / or predicting bone development status, comprising a reagent for detecting sulfate content, wherein the sulfate content serves as a biomarker for bone development status.

2. The kit according to claim 1, wherein The bone development condition is the bone development condition of a mammal; and / or The evaluation and / or prediction of bone development status is for the diagnosis and / or early prediction of bone development disorders and related diseases.

3. The kit according to claim 1, wherein The kit is a kit for detecting sulfate content by spectrophotometry; and / or The kit may further comprise means for obtaining serum and / or plasma samples; and / or The biomarker is the sulfate content in serum and / or plasma.

4. The kit according to any one of claims 1 to 3, wherein The reagents include a standard sulfate aqueous solution with known concentration, anhydrous ethanol and a working reagent.

5. Use of a reagent for detecting sulfate content in the preparation of a kit for evaluating and / or predicting bone development, wherein: The sulfate content serves as a biomarker for bone development status.

6. The use according to claim 5, wherein The bone development condition is the bone development condition of a mammal; and / or The evaluation and / or prediction of bone development status is for the diagnosis and / or early prediction of bone development disorders and related diseases.

7. The use according to claim 5, wherein The kit is a kit for detecting sulfate content by spectrophotometry; and / or The kit may further comprise means for obtaining serum and / or plasma samples; and / or The biomarker is the sulfate content in serum and / or plasma.

8. The use according to claim 5, wherein The reagents include a standard sulfate aqueous solution with known concentration, anhydrous ethanol and a working reagent.

9. The use according to any one of claims 5 to 8, wherein The skeletal development disorder and its related diseases are selected from one or more of short stature, constitutional growth retardation, osteochondrodysplasia and psychosocial dwarfism.

10. A method for evaluating and / or predicting skeletal development for non-diagnostic purposes, comprising the following steps: (1) Determine the sulfate content in samples from subjects and healthy controls, and record them as measured values ​​and reference values; (2) Using the sulfate content as a biomarker for bone development status, and comparing the measured value with a reference value to evaluate and / or predict the bone development status.

11. The method according to claim 10, wherein: The bone development condition is the bone development condition of a mammal; and / or The method detects sulfate content by spectrophotometry; and / or The sample is serum and / or plasma; and / or The method uses the kit according to claim 1 for determination.

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