CN113466469A - Biomarker for detecting autism, detection kit and detection system - Google Patents
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Abstract
The invention provides a biomarker for detecting the severity of autism, namely the level of beta-methylamino-L-alanine (BMAA) in serum, so that the grading evaluation of the severity of autism does not depend on subjective experience judgment, and the diagnosis accuracy is greatly improved; the invention also provides a reagent combination for detecting the level of beta-methylamino-L-alanine (BMAA) in serum, a kit for detecting autism and application thereof; a system for testing a biological sample from an autistic patient to determine the severity of autism in the patient, by which the severity of autism can be graded.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to a biomarker for detecting autism, a detection kit and a detection system.
Background
Autism (autism), also known as autism or autism disorder (autistic disorder), and the like, are representative diseases of Pervasive Developmental Disorder (PDD). DSM-IV-TR classifies PDD into 5 types: autistic disorder, Retts syndrome, childhood disorganized disorder, asperger's syndrome, and unspecified PDD. Among them, autistic disorder and asperger syndrome are common. The prevalence rates of autism are reported differently, generally considered to be about 2-5/ten thousand of children, with a ratio of about 3-4: 1 for men and women, and 3-4 times more for boys than for girls.
Autism is classified into true autism and pseudo autism. True autism (organic autism) is the loss, loss or severe loss of mental function in the brain of a patient due to genetic mutation. Their facies is not different from that of normal people, but their congenital deficiency summarizes, induces, analyzes and judges logic thinking ability and lifelong mental retardation. Pseudo-autism (functional autism) refers to the condition that the brain thought area of the child has no organic lesions and has normal thinking ability, but the loss of intelligence is caused by the imbalance of the development of certain ability in the acquired period. There is a fundamental difference between pseudo-autism and true autism. The true autism is caused by gene mutation, the proportion of the human true autism obtained by calculation is close to one fifteen ten-thousandth, and the true autism children born in China per year are calculated to be not more than 120, and the number of the true autism patients born in the United states per year is lower than 36. True autism should be a rare disease. At present, no effective rehabilitation method exists. Pseudo autism is due to an imbalance in the development of several elements that affect human learning. That is, a patient with extremely weak or over-strong ability may lose some or all of his or her mental abilities, resulting in lifelong intellectual disability and manifesting as the trait of true autism. According to the diagnosis and statistics of domestic hospitals, the incidence rate of autism is 2-3%, wherein the true autism accounts for less than 0.2%, and the false autism accounts for more than 99.8%.
In the 80's of the 20 th century, studies on autism were in a completely new phase. The hypothesis of etiology, called "improper parental regimen", was abandoned by people, exploring the etiology of autism from the biological field, and separating it completely from schizophrenia in terms of identification of clinical symptoms and clinical diagnosis. The Kolvin study showed that autism was not associated with adult psychotic disorders, especially adult schizophrenia. The childhood autism was first identified as a pervasive developmental disorder by DSM-III, published in 1980. Later, with the intensive research on autism, it is gradually recognized that autism is a pervasive development disorder disease of central nervous system caused by the stimulation of various environmental factors under the action of certain genetic factors. On the basis of this knowledge, various studies from molecular inheritance to neuroimmunity, functional imaging, neuroanatomy, neurochemistry and the like have been carried out, and attempts have been made to find the causative factor of autism from these studies. There is no hypothesis that the cause of autism can be completely explained.
The current common method for diagnosing autism is to use a pediatric autism rating scale for diagnosis. The table was compiled in 1980 by e.schopper, r.j.reichler and b.r.renner. The childhood autism rating scale scores fifteen aspects of a child's interpersonal relationships, impersonations (words and actions), emotional responses, physical application abilities, relationships with inanimate objects, adaptation to environmental changes, visual responses, auditory responses, near sensory responses, anxiety responses, verbal communication, non-verbal communication, activity levels, mental functions, and overall impressions based on the child's performance. The scoring criteria were as follows: the total score is less than 30 points: no autism; 30-60 minutes: autism exists; wherein 30-37 minutes: mild to moderate autism; 37-60 min: severe autism.
The autism is diagnosed by using the autism rating scale for children, and the method mainly depends on subjective experience judgment, so that the accuracy of diagnosis is influenced, and the grading evaluation of the autism severity is influenced.
Disclosure of Invention
To overcome the deficiencies of the prior art, it is an object of the present invention to provide biomarkers for the detection of autism, characterized by comprising the level of β -methylamino-L-alanine (BMAA) in serum.
One of the objects of the present invention is to provide the use of said biomarkers for the detection of autism in a system for detecting a biological sample from an autism patient.
One of the purposes of the invention is to provide the application of a reagent combination for detecting the level of beta-methylamino-L-alanine (BMAA) in serum in preparing a kit for detecting autism.
Preferably, the combination of reagents for detecting the level of β -methylamino-L-alanine (BMAA) in serum comprises an ELISA detection reagent;
the ELISA detection reagent comprises phosphate buffer solution of goat anti-rabbit antibody; lyophilizing BMAA calibrator/standard;
rabbit anti-BMAA antibody solution; HRP-labeled BMAA solution; washing liquid; TMB color development liquid; and (4) stopping the solution.
Preferably, the use of the kit for detecting autism comprises the steps of:
obtaining a blood sample from a test subject;
centrifuging a blood sample to obtain serum;
the serum is subjected to ELISA detection, and the severity of autism is determined based on the level of beta-methylamino-L-alanine (BMAA) contained in the serum of the subject as a result of the ELISA detection.
Preferably, the step of centrifuging the blood sample to obtain serum comprises the following steps: collecting venous blood of a tested individual by 3ml, centrifuging at 3000rpm for 5 minutes, and taking supernatant as a serum sample;
the ELISA detection of the serum is carried out, and the severity of the autism is determined according to the level of beta-methylamino-L-alanine (BMAA) contained in the serum of the tested individual according to the result of the ELISA detection, which comprises the following steps:
the method comprises the following steps: adding PBS containing goat anti-rabbit antibody of 1 mug/ml into an ELISA reaction plate, and carrying out sealed incubation for 16 hours at 4 ℃; patting dry the reaction plate, washing the plate hole with PBS containing 0.2% Tween-20, patting dry the reaction plate, and obtaining the coated ELISA plate;
step two: the freeze-dried BMAA calibrator/standard was dissolved in purified water, and the concentrations of the diluted BMAA calibrator/standard were 0ppb, 5ppb, 25ppb, 100ppb, 250ppb, and 500ppb, respectively.
Step three: the following solutions were added to the coated ELISA plate wells: 100 μ l BMAA reference/standard solution or 100 μ l serum sample solution, 50 μ l BMAA-HRP binding solution, 50 μ l rabbit anti-BMAA antibody solution. At least 2 replicate wells per BMAA reference/standard solution or sample;
step four: after mixing for 30 seconds, incubating for 90 minutes at room temperature in the dark;
step five: the contents of the wells were decanted and each well was washed with at least 250 μ l of 0.2% tween-20 in PBS, patted dry and repeated four times. After washing the plate, adding 150 mu l of TMB color developing solution into each reaction plate hole for color development, uniformly mixing for 30 seconds, placing at room temperature in a dark place, keeping the temperature for 30min, and then adding 100 mu l of stop solution into each hole to terminate the reaction;
step six: within 15 minutes after the reaction is stopped, placing the reaction plate in an enzyme-linked immunosorbent assay, and reading an OD450 value;
step seven: drawing a standard curve, and calculating the concentration of the BMAA of the sample according to the standard curve;
step eight: a graded assessment of the severity of autism was made based on the concentration of BMAA in the sample.
It is an object of the present invention to provide a kit for diagnosing autism comprising reagents suitable for detecting the biomarkers of claim 1.
Preferably, the reagents suitable for detecting the biomarkers of claim 1 comprise ELISA detection reagents;
the ELISA detection reagent comprises phosphate buffer solution of goat anti-rabbit antibody; lyophilizing BMAA calibrator/standard;
rabbit anti-BMAA antibody solution; HRP-labeled BMAA solution; washing liquid; TMB color development liquid; and (4) stopping the solution.
Preferably, the use method of the kit for diagnosing autism comprises the following steps:
obtaining a blood sample from a test subject;
centrifuging a blood sample to obtain serum;
the serum is subjected to ELISA detection, and the severity of the patient's autism is determined based on the level of beta-methylamino-L-alanine (BMAA) contained in the serum of the patient as a result of the ELISA detection.
Preferably, the step of centrifuging the blood sample to obtain serum comprises the following steps: venous blood was collected at 3ml, centrifuged at 3000rpm for 5 minutes, and the supernatant was taken as a serum sample.
The ELISA detection of the serum is carried out, and the severity of the patient autism is determined according to the level of beta-methylamino-L-alanine (BMAA) contained in the serum of the patient according to the result of the ELISA detection, which comprises the following steps:
the method comprises the following steps: adding PBS containing goat anti-rabbit antibody of 1 mug/ml into an ELISA reaction plate, and carrying out sealed incubation for 16 hours at 4 ℃; patting dry the reaction plate, washing the plate hole with PBS containing 0.2% Tween-20, patting dry the reaction plate, and obtaining the coated ELISA plate;
step two: dissolving the freeze-dried BMAA calibrator/standard with purified water, and diluting the BMAA calibrator/standard to obtain concentrations of 0ppb, 5ppb, 25ppb, 100ppb, 250ppb and 500ppb respectively;
step three: the following solutions were added to the coated ELISA plate wells: 100 μ l BMAA reference/standard solution or 100 μ l sample solution, 50 μ l BMAA-HRP binding solution, 50 μ l BMAA antibody solution. At least 2 replicate wells per BMAA reference/standard solution or sample;
step four: after mixing for 30 seconds, incubating for 90 minutes at room temperature in the dark;
step five: the contents of the wells were decanted and each well was washed with at least 250 μ l of 0.2% tween-20 in PBS, patted dry and repeated four times. After washing the plate, adding 150 mu l of TMB color developing solution into each reaction plate hole for color development, uniformly mixing for 30 seconds, placing at room temperature in a dark place, keeping the temperature for 30min, and then adding 100 mu l of stop solution into each hole to terminate the reaction;
step six: within 15 minutes after the reaction is stopped, placing the reaction plate in an enzyme-linked immunosorbent assay, and reading an OD450 value;
step seven: drawing a standard curve, and calculating the concentration of the BMAA of the sample according to the standard curve;
step eight: a graded assessment of the severity of autism was made based on the concentration of BMAA in the sample.
It is an object of the present invention to provide a system for testing a biological sample from an autistic patient to determine the severity of autism in the patient, comprising:
a blood processing device for centrifuging a sample to be tested, which is taken from a test subject, to obtain serum;
the ELISA detection device is connected with the blood treatment device and is used for detecting an enzyme-labeled value of the serum treated by the ELISA detection reagent; and
and the analysis device is connected with the ELISA detection device, performs concentration analysis on the biomarker contained in the ELISA detection device based on the enzyme label value, and judges the severity of the autism of the tested individual of the sample to be tested according to the analysis result.
Preferably, the blood processing apparatus operating steps include: venous blood was collected at 3ml, centrifuged at 3000rpm for 5 minutes, and the supernatant was taken as a serum sample.
The ELISA detection device and the analysis device comprise the following operation steps:
the method comprises the following steps: adding PBS containing goat anti-rabbit antibody of 1 mug/ml into an ELISA reaction plate, and carrying out sealed incubation for 16 hours at 4 ℃; patting dry the reaction plate, washing the plate hole with PBS containing 0.2% Tween-20, patting dry the reaction plate, and obtaining the coated ELISA plate;
step two: the lyophilized BMAA calibrator/standard was dissolved with purified water and the concentrations of the diluted BMAA calibrator/standard were 0, 5, 25, 100, 250, 500(ppb), respectively.
Step three: the following solutions were added to the coated ELISA plate wells: 100 μ l BMAA reference/standard solution or 100 μ l sample solution, 50 μ l BMAA-HRP binding solution, 50 μ l BMAA antibody solution. At least 2 replicate wells were made per BMAA reference/standard solution or sample.
Step four: after mixing for 30 seconds, incubation was carried out for 90 minutes at room temperature in the dark.
Step five: the contents of the wells were decanted and each well was washed with at least 250 μ l of 0.2% tween-20 in PBS, patted dry and repeated four times. After washing the plate, adding 150 mu l of TMB color developing solution into each reaction plate hole for color development, uniformly mixing for 30 seconds, placing at room temperature in a dark place, keeping the temperature for 30min, and then adding 100 mu l of stop solution into each hole to terminate the reaction;
step six: within 15 minutes after the termination of the reaction, the reaction plate was placed in a microplate reader and the OD450 value was read.
Step seven: and drawing a standard curve, and calculating the concentration of the BMAA of the sample according to the standard curve.
Step eight: a graded assessment of the severity of autism was made based on the concentration of BMAA in the sample.
In this patent, the term "biomarker" is to be understood in a broad sense and includes any detectable biomarker capable of reflecting an abnormal condition, which may include genetic markers, species markers (species/genus markers) and functional markers (KO/OG markers), which have a very broad range of uses, either for disease diagnosis, for determining disease stage, or for evaluating the safety and effectiveness of new drugs or therapies in a target population.
In addition, in the present invention, the agent for detecting or measuring the level of BMAA is an antibody, and by using an immunological method based on an antigen-antibody reaction, it is possible to detect or measure the level of BMAA. Examples of the Assay method used for this purpose include western blotting, enzyme linked immunosorbent Assay (ELlSA), Radioimmunoassay (RIA), Radioimmunoassay (radioimmunodiffusion), Ouchtery (Ouchterlony) immunodiffusion, rocket (rocket) immunoelectrophoresis, tissue immunostaining, Immunoprecipitation Assay (Immunoprecipitation Assay), Complement Fixation Assay (complementary hybridization Assay), Fluorescence Activated Cell Sorter (FACS), protein chip (protein chip), etc., and these are merely illustrative of antibody-antigen immunoreaction, and the present invention is not limited to these.
The invention has the following beneficial effects:
in the process of researching the relevance between the autism and the intestinal microorganisms, the inventor finds that the prokaryote Cyanobacteria existing in the intestinal microorganisms has obvious relevance to the occurrence of the autism, which indicates that the prokaryote Cyanobacteria has close relation to the occurrence of the autism. The invention discovers that the concentration of beta-methylamino-L-alanine (BMAA) has obvious correlation with the severity of autism, and the higher the concentration of the BMAA is, the more serious the autism is. It is suggested that BMAA may be a significant cause of autism. The invention takes the BMAA content in the serum as a detection marker for grading the severity of the autism, determines the severity of the autism by detecting the concentration of the BMAA, changes the currently adopted method for diagnosing the autism by depending on an autism rating scale and determining the severity of the autism, overcomes the defect that the diagnosis of the grading of the severity of the autism depends on subjective experience judgment to a greater extent, and greatly improves the diagnosis accuracy.
The detection time of the kit provided by the invention is only about 3 hours, and only venous blood sampling is needed for patients with autism. Compared with the existing autism detection technology, the ELISA detection kit has the characteristics of objectivity, simplicity, convenience, quickness and the like, reduces the participation time of autism patients, and greatly reduces the working difficulty of medical workers.
Furthermore, compared with the method for diagnosing the autism and determining the severity of the autism by depending on the autism rating scale, the method for detecting the concentration of the BMAA has high sensitivity, and can detect even slight change of the concentration of the BMAA, so the method has wide application in future, such as clinical evaluation of the curative effect of the autism treatment, dynamic monitoring of the state of illness of an autistic patient and the like.
Drawings
FIG. 1 is a diagram showing ROC curve analysis of ELISA kit of the present invention for detecting and grading the severity of autism.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press, 1989), or according to the manufacturer's recommendations. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
In order to evaluate whether the concentration of BMAA in serum can be used as an evaluation factor of the severity of autism, the kit provided by the invention collects a blood sample of an autism patient, performs an ELISA experiment to detect the concentration of BMAA in serum of the autism patient, evaluates the grading condition of the severity of autism of the patient according to the concentration, and compares the grading condition with a diagnosis result of a doctor through a children autism evaluation scale to find that the consistency of the results of the two reaches 90%, so that the kit provided by the invention can be used for grading evaluation of the severity of autism instead of the children autism evaluation scale.
Example 1
In this example, blood samples from patients with autism were analyzed by ELISA and then statistically analyzed to determine the concentration of BMAA as a biomarker for assessing whether or not autism is present and the severity of the condition. The method comprises the following specific steps:
1. sample source
Sample sources and inclusion criteria were as follows:
patients diagnosed with autism. Exclusion criteria: (ii) suffering from other psychiatric disorders (such as schizophrenia) and other neurodevelopmental disorders; ② suffering from hereditary metabolic diseases; history of serious physical diseases such as serious neurological diseases and craniocerebral injury history;
the study was divided into two phases: the first phase is to perform a comparative study of the consistency of the diagnostic results of the present invention with the autism rating scale, and the second phase is to perform a comparative study of the sensitivity of the present invention with the method of diagnosing the autism rating scale.
In the first stage of study, 10 autistic children were collected, 9 men and 1 woman; the age is 3-7 years;
in the second stage study, 9 children were collected, 6 of autistic children and 3 of children with developmental delay, 6 of autistic children, and 0 of women; the age is 3-7 years. 3 children with hypoevolutism, 1 male and 2 female; the age is 3-7 years.
Blood collection and supernatant acquisition:
about 3ml of venous blood was collected with a disposable blood collection tube, and then centrifuged at 3000rpm for 5 minutes with a centrifuge, and the supernatant was collected as a serum sample.
And (3) ELISA detection:
coating a reaction plate: adding PBS containing goat anti-rabbit antibody of 1 mug/ml into an ELISA reaction plate, and carrying out sealed incubation for 16 hours at 4 ℃; patting dry the reaction plate, washing the plate hole with PBS containing 0.2% Tween-20, patting dry the reaction plate, and obtaining the coated ELISA plate;
add reference/standard solution: 100 μ l of diluted BMAA calibrator/standard was sequentially added to the well-coated wells at concentrations of 0ppb, 5ppb, 25ppb, 100ppb, 250ppb, and 500ppb, respectively. At least two parallel wells were made for each concentration.
Adding a serum sample: to the other well-coated wells of the microplate, 100. mu.l of each serum sample was added in sequence.
Adding a binding solution: add 50. mu.l of BMAA-HRP conjugate to each well.
Adding an antibody solution: add 50. mu.l of BMAA antibody solution to each well.
And (3) incubation: after mixing for 30 seconds, incubation was carried out for 90 minutes at room temperature in the dark.
Washing the plate: the contents of the wells were decanted and each well was washed with at least 250 μ l of 0.2% tween-20 in PBS, patted dry and repeated four times.
Color development and reaction termination: after washing the plate, adding 150 mu l of TMB color developing solution into each reaction plate hole for color development, uniformly mixing for 30 seconds, placing at room temperature in a dark place, keeping the temperature for 30min, and then adding 100 mu l of stop solution into each hole to terminate the reaction;
and (3) detection: within 15 minutes after the termination of the reaction, the reaction plate was placed in a microplate reader and the OD450 value was read.
Calculating BMAA concentration of the sample: and drawing a standard curve of the absorbance of the standard substance at 450nm and the concentration of the BMAA, and calculating the concentration of the BMAA of the sample according to the standard curve.
Grading assessment of the severity of autism: determining the severity of autism of the serum test subject according to the BMAA concentration of the sample.
The kit detects BMAA concentration and autism severity evaluation criteria: the BMAA concentration is less than or equal to 101ppb, and the severity of autism is mild to moderate; BMAA concentration >101ppb, autism severity was severe.
The preparation method of the reagent related to each step is as follows:
PBS: weighing potassium dihydrogen phosphate (KH)2PO4)0.2g of disodium hydrogen phosphate (Na)2HPO4·12H20)2.9g, sodium chloride (NaCl)8.0g, potassium chloride (KCI)0.2g, and water to 1000 mL.
PBS containing 0.2% tween-20: weighing potassium dihydrogen phosphate (KH)2PO4)0.2g of disodium hydrogen phosphate (Na)2HPO4·12H20)2.9g, 8.0g of sodium chloride (NaCl), 0.2g of potassium chloride (KCI), 202 mL of Tween-202, and water to 1000 mL.
Statistical analysis
4.1. The invention analyzes the consistency of the diagnosis result of the autism rating scale
10 patient samples were diagnosed using the kit of the invention and the autism rating scale and the results are shown in Table 1
TABLE 1 diagnosis of 10 patient samples with ELISA kit of the invention and autism rating Scale according to the invention, respectively
The two evaluation methods of the severity of the autism are only inconsistent in 1 case, and the severity of the autism detected by using the ELISA kit provided by the invention is 90% consistent with the diagnosis result of the currently and commonly used autism rating scale.
4.2. Sensitivity analysis of the invention and autism rating Scale diagnosis
The diagnosis of 9 donor samples using the kit of the invention and the autism rating scale is shown in Table 2
TABLE 2 diagnosis of 9 blood samples using the ELISA kit of the present invention and the autism rating Scale, respectively
For the detection of patients with autism, only 1 case of the two methods for evaluating the severity of autism is inconsistent; in addition, although the doctor considers that the development is slow and the autism is not autism as a result of diagnosis of the autism rating scale used in 3 cases, the severity of autism can be determined by the kit of the present invention. It can be seen that the sensitivity of the kit of the invention is greater than that of currently generally used autism rating scales. 3 children with hypoevolutism are also currently carrying out the rehabilitation training of autism, and the visible disease condition and the autism have a plurality of similarities. But if the disease is evaluated as hypoevolutism, the disease may be delayed by the parents' unappreciation. In this regard, it is important that the present invention has higher sensitivity.
4.3. Feasibility and necessity of the invention for diagnosing autism severity degree by replacing autism rating scale
The kit and the autism rating scale are used for carrying out statistical analysis on the diagnosis results of the severity of 16 autism patients, and the results show that the toxin concentration detected by the kit has obvious correlation with the evaluation results of the autism rating scale, the correlation coefficient is 1.00, and the P value is 0.011. The results are shown in Table 3.
ROC curve analysis of the kit autism severity grading result shows that A mu C is 0.857, P value is 0.017 and statistical significance is achieved. The optimal cut-off value is 101.1617, the corresponding specificity is 0.889, and the sensitivity is 0.857 (see figure 1), which indicates that the kit has higher sensitivity and specificity when applied to the detection of the severity of the autism.
The experimental result shows that the BMAA concentration in the serum of the patient has obvious correlation with the severity of the autism, so that the determination of the severity of the autism of the patient by detecting the BMAA concentration in the serum of the patient by using the kit is feasible, and the ROC curve analysis result also shows that the kit has higher sensitivity and specificity when being applied to the detection of the severity of the autism.
Moreover, because the kit adopts an ELISA method to detect the concentration of the BMAA in the serum of the patient, the ELISA sensitivity can reach ng level, so the kit can detect the change of the concentration of the BMAA in the serum of the patient very sensitively, which is very beneficial to the treatment effect of the autism patient and the judgment of the change condition of the self-illness state of the autism patient, and therefore, the kit has strong feasibility and necessity for replacing an autism rating scale to diagnose the severity degree of the autism.
TABLE 3 correlation of the measured toxin concentration of the kit of the present invention with the evaluation of the autism rating scale
Correlation coefficient
*. at a confidence (double test) of 0.05, the correlation was significant.
Claims (10)
1. A biomarker for the detection of autism comprising the level of β -methylamino-L-alanine (BMAA) in serum.
2. Use of a biomarker for the detection of autism according to claim 1 in a system for detecting a biological sample from an autism patient.
3. Use of a combination of reagents for detecting levels of beta-methylamino-L-alanine (BMAA) in serum in the manufacture of a kit for detecting autism.
4. The use of claim 3, wherein the combination of reagents for detecting levels of β -methylamino-L-alanine (BMAA) in serum comprises an ELISA detection reagent; the ELISA detection reagent comprises phosphate buffer solution of goat anti-rabbit antibody; lyophilizing BMAA calibrator/standard; rabbit anti-BMAA antibody solution; HRP-labeled BMAA solution; washing liquid; TMB color development liquid; and (4) stopping the solution.
5. The use according to claim 4, wherein the use of the kit for the detection of autism comprises the steps of:
obtaining a blood sample from a test subject;
centrifuging a blood sample to obtain serum;
the serum is subjected to ELISA detection, and the severity of autism is determined based on the level of beta-methylamino-L-alanine (BMAA) contained in the serum of the subject as a result of the ELISA detection.
6. A kit for diagnosing autism comprising reagents suitable for detecting the biomarkers of claim 1.
7. The kit for diagnosing autism of claim 6, wherein the reagents suitable for detecting the biomarkers of claim 1 comprise ELISA detection reagents; the ELISA detection reagent comprises phosphate buffer solution of goat anti-rabbit antibody; lyophilizing BMAA calibrator/standard; rabbit anti-BMAA antibody solution; HRP-labeled BMAA solution; washing liquid; TMB color development liquid; and (4) stopping the solution.
8. The kit for diagnosing autism according to claim 7, wherein the method of using the kit for diagnosing autism comprises the steps of:
obtaining a blood sample from a test subject;
centrifuging a blood sample to obtain serum;
the serum is subjected to ELISA detection, and the severity of the patient's autism is determined based on the level of beta-methylamino-L-alanine (BMAA) contained in the serum of the patient as a result of the ELISA detection.
9. The kit for diagnosing autism as in claim 8, wherein centrifuging the blood sample to obtain serum comprises the steps of: venous blood was collected at 3ml, centrifuged at 3000rpm for 5 minutes, and the supernatant was taken as a serum sample.
The ELISA detection of the serum is carried out, and the severity of the patient autism is determined according to the level of beta-methylamino-L-alanine (BMAA) contained in the serum of the patient according to the result of the ELISA detection, which comprises the following steps:
the method comprises the following steps: adding PBS containing goat anti-rabbit antibody of 1 mug/ml into an ELISA reaction plate, and carrying out sealed incubation for 16 hours at 4 ℃; patting dry the reaction plate, washing the plate hole with PBS containing 0.2% Tween-20, patting dry the reaction plate, and obtaining the coated ELISA plate;
step two: dissolving the freeze-dried BMAA calibrator/standard with purified water, and diluting the BMAA calibrator/standard to obtain concentrations of 0ppb, 5ppb, 25ppb, 100ppb, 250ppb and 500ppb respectively;
step three: the following solutions were added to the coated ELISA plate wells: 100 μ l BMAA reference/standard solution or 100 μ l sample solution, 50 μ l BMAA-HRP binding solution, 50 μ l BMAA antibody solution. At least 2 replicate wells per BMAA reference/standard solution or sample;
step four: after mixing for 30 seconds, incubating for 90 minutes at room temperature in the dark;
step five: the contents of the wells were decanted and each well was washed with at least 250 μ l of 0.2% tween-20 in PBS, patted dry and repeated four times. After washing the plate, adding 150 mu l of TMB color developing solution into each reaction plate hole for color development, uniformly mixing for 30 seconds, placing at room temperature in a dark place, keeping the temperature for 30min, and then adding 100 mu l of stop solution into each hole to terminate the reaction;
step six: within 15 minutes after the reaction is stopped, placing the reaction plate in an enzyme-linked immunosorbent assay, and reading an OD450 value;
step seven: drawing a standard curve, and calculating the concentration of the BMAA of the sample according to the standard curve;
step eight: a graded assessment of the severity of autism was made based on the concentration of BMAA in the sample.
10. A system for testing a biological sample from an autistic patient to determine the severity of autism in the patient, comprising:
a blood processing device for centrifuging a sample to be tested, which is taken from a test subject, to obtain serum;
the ELISA detection device is connected with the blood treatment device and is used for detecting an enzyme-labeled value of the serum treated by the ELISA detection reagent; and
and the analysis device is connected with the ELISA detection device, performs concentration analysis on the biomarker contained in the ELISA detection device based on the enzyme label value, and judges the severity of the autism of the tested individual of the sample to be tested according to the analysis result.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1860369A (en) * | 2003-08-12 | 2006-11-08 | 种族医学学会 | Neurotoxic amino acid or neurotoxic derivative thereof associated with neurological disorders |
| CN103308639A (en) * | 2013-05-17 | 2013-09-18 | 南京大学 | Detection method of content of blue algae toxin beta-methylamino-L-alanine in aquatic products |
| CN107058560A (en) * | 2017-05-04 | 2017-08-18 | 深圳市英马诺生物科技有限公司 | Self-closing disease biomarker and its detection kit, application |
| CN107167610A (en) * | 2017-05-04 | 2017-09-15 | 深圳市英马诺生物科技有限公司 | Self-closing disease biomarker and its detection kit |
| US20200157625A1 (en) * | 2017-03-21 | 2020-05-21 | Quadrant Biosciences Inc. | Analysis of autism spectrum disorder |
| US20210164051A1 (en) * | 2019-12-02 | 2021-06-03 | The Institute for Ethnomedicine dba Brain Chemistry Labs | Methods of detection and analysis of nucleic acid in neural-derived exosomes |
-
2021
- 2021-07-07 CN CN202110765346.0A patent/CN113466469B/en active Active
- 2021-07-07 CN CN202310445987.7A patent/CN116593706A/en not_active Withdrawn
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1860369A (en) * | 2003-08-12 | 2006-11-08 | 种族医学学会 | Neurotoxic amino acid or neurotoxic derivative thereof associated with neurological disorders |
| CN103308639A (en) * | 2013-05-17 | 2013-09-18 | 南京大学 | Detection method of content of blue algae toxin beta-methylamino-L-alanine in aquatic products |
| US20200157625A1 (en) * | 2017-03-21 | 2020-05-21 | Quadrant Biosciences Inc. | Analysis of autism spectrum disorder |
| CN107058560A (en) * | 2017-05-04 | 2017-08-18 | 深圳市英马诺生物科技有限公司 | Self-closing disease biomarker and its detection kit, application |
| CN107167610A (en) * | 2017-05-04 | 2017-09-15 | 深圳市英马诺生物科技有限公司 | Self-closing disease biomarker and its detection kit |
| US20210164051A1 (en) * | 2019-12-02 | 2021-06-03 | The Institute for Ethnomedicine dba Brain Chemistry Labs | Methods of detection and analysis of nucleic acid in neural-derived exosomes |
Non-Patent Citations (4)
| Title |
|---|
| ANTHONY LAUGERAY等: "Perinatal Exposure to the Cyanotoxin β-N-Méthylamino-L-Alanine (BMAA) Results in Long-Lasting", 《NEUROTOX RES》 * |
| ANTHONY LAUGERAY等: "Perinatal Exposure to the Cyanotoxin β-N-Méthylamino-L-Alanine (BMAA) Results in Long-Lasting", 《NEUROTOX RES》, vol. 33, 6 September 2017 (2017-09-06), pages 87 - 112, XP036379748, DOI: 10.1007/s12640-017-9802-1 * |
| 周珂新等: "阿尔茨海默病与肠道菌群的关系及菌群调节对其防治的展望", 《中国药理学与毒理学杂志》 * |
| 周珂新等: "阿尔茨海默病与肠道菌群的关系及菌群调节对其防治的展望", 《中国药理学与毒理学杂志》, no. 11, 15 November 2016 (2016-11-15), pages 1198 - 1205 * |
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Application publication date: 20211001 Assignee: Beijing Changshunning Biotechnology Co.,Ltd. Assignor: Beijing Changyou Biotechnology Co.,Ltd. Contract record no.: X2024980006639 Denomination of invention: Biomarkers, testing kits, and testing systems for autism detection Granted publication date: 20240116 License type: Exclusive License Record date: 20240603 |