CN109682969A - A kind of liquid phase chip reagent box detecting intractable epilepsy disease - Google Patents

A kind of liquid phase chip reagent box detecting intractable epilepsy disease Download PDF

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Publication number
CN109682969A
CN109682969A CN201910120115.7A CN201910120115A CN109682969A CN 109682969 A CN109682969 A CN 109682969A CN 201910120115 A CN201910120115 A CN 201910120115A CN 109682969 A CN109682969 A CN 109682969A
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detection antibody
antibody
microballoon
detection
coated
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洪桢
周东
屈波
王锐
屈珏
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West China Hospital of Sichuan University
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2446/00—Magnetic particle immunoreagent carriers
    • G01N2446/80—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids
    • G01N2446/84—Polymer coating, e.g. gelatin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2857—Seizure disorders; Epilepsy

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Abstract

The invention discloses a kind of liquid phase chip reagent box for detecting intractable epilepsy disease, kit includes coating microballoon, the detection antibody of biotin labeling, streptomysin-R phycoerythrin, reaction buffer and reaction dilution；Wherein, coating microballoon includes the microballoon of the microballoon for being coated with TIMP-1 capture antibody, the microballoon of coating MMP2 capture antibody, the microballoon of coating MMP3 capture antibody and coating MMP9 capture antibody, and there is the microballoon for being coated with different capture antibody different colors to encode；Detection antibody includes TIMP-1 detection antibody, MMP2 detection antibody, MMP3 detection antibody and MMP9 detection antibody；Reaction buffer is weakly alkaline PBS buffer solution；Dilution buffer is the PBS buffer solution for including 0.05wt%~0.15wt%BSA, and pH value is 7.0~7.5.Using the kit in the present invention, the purpose of discovery and Accurate Diagnosis epilepsy in time can achieve.

Description

Liquid chip kit for detecting intractable epilepsy
Technical Field
The invention belongs to the technical field of medical biology, and particularly relates to a liquid chip kit for detecting intractable epilepsy.
Background
Epilepsy is a chronic recurrent transient cerebral dysfunction syndrome, characterized by recurrent epileptic seizures caused by highly synchronized abnormal firing of brain neurons. Epilepsy is one of the common diseases of the nervous system, and the prevalence rate is second to stroke. The incidence of epilepsy is age-related, and generally considered to be the highest in prevalence within 1 year of age, and gradually decreases after 1-10 years of age. The ratio of the male to the female of epileptic patients in China is 1.15-1.7: 1.
Refractory epilepsy is also called refractory epilepsy and generally refers to progressive diseases or space occupying pathological changes of the central nervous system, but clinical persistence is achieved, after more than 2 years of formal antiepileptic treatment, the trial of main antiepileptic drugs is carried out singly or together, the maximum dose which can be tolerated by a patient is achieved, the blood concentration reaches an effective range, the seizure cannot be controlled, the daily life is influenced, and the refractory epilepsy can be determined to be refractory epilepsy which accounts for about 20-30% of patients with epilepsy. The intractable epilepsy has various causes, and most of the intractable epilepsy has brain nourishing lesions, such as hippocampal sclerosis, brain trauma, tumor, nodular sclerosis, congenital dysplasia and the like. A rapid and accurate diagnosis of epilepsy is of great importance to clinicians taking relevant therapeutic measures or decisions for a patient.
LaFrance et al found that TIMP-1 was lower in serum than normal persons and expected to become serum biomuraker for epilepsy, but it was only useful in small sample experiments, and there was no significant difference between TIMP-1 level in serum of large sample epilepsy patients and normal group. In addition, there have been reports that indicators such as HSP70, S100B, NSE and the like have higher serum levels than normal persons in intractable epilepsy patients, but all of them have problems such as poor reproducibility and reliability of results due to unstable detection methods and poor signal-to-noise ratios of detection methods, and thus cannot be applied in large scale. The feasibility of MicroRNA as a diagnostic marker of epilepsy is also researched and reported, but due to the reasons of small sample size, special requirements of a detection method, high cost and the like, the reliability, clinical significance and popularization of the result are subject to further examination.
The content of MMPs in brain tissues and cerebrospinal fluid of epileptics is obviously higher than that of normal people, the MMPs are an endopeptidase family containing zinc ions, 23 family members are separated out in total, and the serial numbers of the family members are respectively MMP 1-23. The activity of MMPs can be regulated by Tissue Inhibitor of Metalloproteinases (TIMPs), and under normal physiological conditions, MMPs and TIMPs jointly regulate the renewal of extracellular matrix (ECM), maintain the stability of blood brain barrier, cell membrane and cells, but if MMPs/TIMPs are unbalanced, the structure of central nervous system, neurochemical transmitters and channels influencing cell regeneration and death will be changed, thereby generating the basis of chronic epilepsy. However, high-level studies of MMPs as diagnostic markers are still insufficient at home and abroad.
Disclosure of Invention
Aiming at the prior art, the invention provides a liquid chip kit for detecting intractable epilepsy, so as to achieve the purpose of timely finding and accurately diagnosing epilepsy.
In order to achieve the purpose, the invention adopts the technical scheme that: providing a liquid chip kit for detecting intractable epilepsy, which comprises coated microspheres, a biotin-labeled detection antibody, streptomycin, a reaction buffer solution and a reaction diluent; wherein,
the coated microspheres comprise microspheres coated with TIMP-1 capture antibody, microspheres coated with MMP2 capture antibody, microspheres coated with MMP3 capture antibody and microspheres coated with MMP9 capture antibody, and the concentration of each microsphere is 3 x 106～8×106Per mL, and the microspheres coated with different capture antibodies have different color codes;
the detection antibody comprises a TIMP-1 detection antibody, an MMP2 detection antibody, an MMP3 detection antibody and an MMP9 detection antibody, and the concentration of each detection antibody is 0.08-0.12 mu g/mL;
the reaction buffer solution is a PBS buffer solution with the pH value of 7.0-7.5;
the dilution buffer is PBS buffer containing 0.05 wt% -0.15 wt% BSA, and the pH value is 7.0-7.5.
On the basis of the technical scheme, the invention can be further improved as follows.
Further, the concentration of each coated microsphere in the kit is 5 × 106one/mL.
Further, the concentration of each detection antibody in the kit was 0.1. mu.g/mL.
Further, the mass fraction of BSA in the dilution buffer was 0.1 wt%.
The invention has the beneficial effects that: the liquid chip kit has high sensitivity, greatly improves the accuracy of early diagnosis of intractable epilepsy by combined detection of a plurality of markers, has quick detection and good stability, can realize high-throughput detection, and has low cost.
Drawings
FIG. 1 is a standard curve of each detection index in the kit, wherein A is a standard curve of TIMP-1; b is the standard curve for MMP 2; c is a standard curve for MMP 3; d is a standard curve for MMP 9;
FIG. 2 is a liquid phase chip technology for detecting the MMP-2 content in serum;
FIG. 3 is a graph showing serum MMP-2 levels in epileptic patients and normal controls;
FIG. 4 is a liquid phase chip technology for detecting the MMP-3 content in serum;
FIG. 5 is a graph showing serum MMP-3 levels in epileptic patients and normal controls;
FIG. 6 is a liquid phase chip technique for detecting the TIMP-1 content in serum;
FIG. 7 is a ROC curve using serum MMP-2 levels as a diagnostic marker for epileptic and normal controls;
FIG. 8 is a ROC curve using serum MMP-3 levels as a diagnostic marker for epileptic and normal controls;
FIG. 9 is a ROC curve using serum TIMP-1 levels as a diagnostic marker for epileptic patients and normal controls.
Detailed Description
The following examples are provided to illustrate specific embodiments of the present invention.
EXAMPLE one preparation of liquid phase chip
1. Preparation of coated microspheres
(1) The microsphere suspension (magnetic microspheres, bio-rad) was vortexed and sonicated sequentially for about 20 seconds, followed by 250. mu.L (about 5X 10)6Microspheres) in a clean centrifuge tube (Axygen, usa) and labeled with the microsphere type number and protein name; then putting the mixture into a centrifuge, separating the mixture for 4min at room temperature of 14000g, and then sucking supernatant;
(2) add 100. mu.L ddH to centrifuge tubes2O, sequentially carrying out vortex and ultrasonic treatment for about 20s, separating at room temperature of 14000g for 4min, and sucking supernatant;
(3) 160. mu.L of NaH at pH 6.2 and 0.1M was added to the centrifuge tube2PO4Sequentially vortexing and sonicating for about 20S, then adding 20 mu L N-hydroxy thiosuccinimide (S-NHS), vortexing and mixing uniformly, then adding 20 mu L of 1-ethyl- (3-dimethylaminopropyl) carbonyl diimine hydrochloride (EDC), and sequentially vortexing and sonicating for about 20S; then carrying out rotary incubation for 20min at room temperature in the dark (if the microspheres are not in a suspended state, vortex once every 10 min), then separating for 4min at 14000g, and sucking out the supernatant;
(4) adding 250 mu LpH of 5.0 and 0.05M 2- (N-morpholino) ethanesulfonic acid (MES) into the system treated in the step (3), sequentially performing vortex and ultrasonic treatment for about 20s, separating for 4min at 14000g, and sucking out the supernatant;
(5) resuspending the microspheres in 50. mu.L of MES at pH 5.0 and concentration 0.05M, vortexing and sonicating for about 20 s;
(6) adding 25 μ g of capture antibody into the microsphere, adding MES with pH of 5.0 and concentration of 0.05M to a final volume of 500ul, respectively vortexing and sonicating for about 20s, incubating for 2h at room temperature in a dark place, separating for 4min at 14000g, and removing supernatant;
(7) adding 1ml PBS buffer solution into the system treated in the step (6), rotationally incubating for 30min at room temperature in a dark place, separating for 4min at 14000g, and sucking supernatant; marking the microsphere concentration and the coupling time on a centrifuge tube, and then placing the centrifuge tube for storage at 4 ℃ in a dark place to finish the preparation of the coated microspheres.
According to the method, 3 types of capture antibody coated microspheres are prepared, which respectively comprise the following steps: 34# microsphere coated with TIMP-1 capture antibody, 45# microsphere coated with MMP2 capture antibody, and 26# microsphere coated with MMP9 capture antibody.
2. Construction of chip kit
When the kit is constructed, 3 prepared coated microspheres are uniformly mixed and then put into the kit, and the concentration of each coated microsphere is ensured to be the same and is 3 multiplied by 106～8×106In the range of one/mL; in addition, a detection antibody, streptomycin-R phycoerythrin, a reaction buffer solution, a dilution buffer solution, a sealing film, a 96-well plate and the like are also arranged in the reagent box; the detection antibodies in the kit comprise a TIMP-1 detection antibody, an MMP2 detection antibody, an MMP3 detection antibody and an MMP9 detection antibody, wherein each detection antibody is marked by biotin, and the concentration of each detection antibody is 0.08-0.12 mu g/mL; the reaction buffer solution in the kit is a PBS buffer solution with the pH value of 7.0-7.5, the dilution buffer solution is a PBS buffer solution containing 0.05 wt% -0.15 wt% BSA, and the pH value is 7.0-7.5.
Example II use of a kit for detecting intractable epilepsy
1. Detection method
(1) Before use, all the reagents are taken out from the reagent box and are placed to room temperature;
(2) taking out hippocampal tissues of a rhesus monkey suffering from epilepsy and a healthy rhesus monkey, respectively placing the hippocampal tissues on ice to melt, uniformly mixing by vortex, and taking a 96-well plate to dilute the hippocampal tissues by 20 times;
(3) preparing TIMP-1, MMP2, MMP3 and MMP9 standard proteins, respectively marking 7 EP tubes with S1, S2, S3, S4, S5, S6 and S7, respectively, performing 2-fold serial dilution, and uniformly mixing by vortex after each standard protein is diluted and replacing a gun head;
(4) taking a 96-well plate, and opening a plate washer-Prime-rinse (channel2) -Prime-place plate-Run 5: MAGX 3;
(5) taking prepared capture antibody coated microspheres, mixing uniformly for 20s by vortex, performing ultrasonic treatment for 20s, adding 2500 microspheres in each hole into 1% PBS, mixing uniformly for 20s by vortex, performing ultrasonic treatment for 20s, and setting the final volume to be 10 mu L/hole;
(6) immediately adding 20 times diluted hippocampal tissue and serial diluted standard protein (adding hippocampal tissue or standard protein with different dilution times to different wells of antibody), each 30 μ L/well;
(7) wrapping a 96-well plate with aluminum foil paper, and performing shaking incubation reaction for 60min at room temperature;
(8) three washes with PBS buffer including 0.1 wt% BSA, Run 5: MAGX 3;
(9) diluting biotin-labeled detection antibodies (biotin-labeled TIMP-1 detection antibodies, MMP2 detection antibodies, MMP3 detection antibodies and MMP9 detection antibodies) with PBS buffer solution with the pH of 7.0-7.5, wherein the concentration of the detection antibodies is 50 mu L/hole;
(10) wrapping a 96-well plate with aluminum foil paper, and performing shaking incubation reaction for 60min at room temperature;
(11) three washes with PBS buffer including 0.1 wt% BSA, each antibody concentration 0.1 μ g/mL, Run 5: MAGX 3;
(12) diluting streptomycin-R phycoerythrin (1: 500) with PBS buffer solution with pH of 7.0-7.5, 50 muL/hole;
(13) wrapping a 96-well plate with aluminum foil paper, and performing shaking incubation reaction for 60min at room temperature;
(14) three washes with PBS buffer including 0.1 wt% BSA, Run 5: MAGX 3;
(15) resuspending the microspheres in 100 μ L/well of 1% PBS, shaking at room temperature for 3-5 min;
(16) and reading the result on a Luminex series liquid phase chip analyzer, wherein the analyzer can automatically draw a standard curve and calculate the measured value of the sample to be measured.
2. Analysis of results
FIG. 1 is a standard curve of each detection index in the kit of the present invention. FIGS. 2 and 3 are schematic diagrams of detection of MMP-2 content in serum by LC chip technique and serum MMP-2 levels in epileptic and normal controls, respectively; as can be seen from FIGS. 2 and 3 (CTL is normal control; EP is epileptic group), serum MMP-2 levels decreased with age in either epileptic or normal control, and serum MMP-2 levels were significantly lower in epileptic than in normal control. FIGS. 4 and 5 are schematic diagrams of detection of MMP-3 content in serum by LC chip technique and serum MMP-3 levels in epileptic and normal controls, respectively; as can be seen from FIGS. 4 and 5, serum MMP-3 levels in epileptic patients increased with age (P <0.05), and serum MMP-3 levels were significantly lower in epileptic patients than in the normal control group, and female MMP-3 serum levels were significantly lower than in males, with a statistical difference (P < 0.05). FIG. 6 shows that the liquid phase chip technology detects the TIMP-1 content in serum, and the TIMP-1 level in serum has no obvious correlation with age (P > 0.05).
FIG. 7 and FIG. 8 are ROC curves for MMP-2 and MMP-3 for epileptic patients and normal controls of different ages, respectively, from which it can be seen that, when the concentration is 175.40ng/ml, the sensitivity of serum MMP-2 as diagnostic indicator for epilepsy and normal controls is 71.13% and the specificity is 62.66%; in the age group of 20-40 years, when the serum MMP-3 concentration of men and women is 23.87ng/ml and 12.31ng/ml respectively, the diagnostic sensitivity is 72.22% and 45%, respectively, and the specificity is 76.67% and 94.12%, respectively; in the age group over 40 years, when the serum MMP-3 concentration of men and women was 20.70ng/ml and 10.92ng/ml, respectively, the diagnostic sensitivity was 85.71% and 85.62%, respectively, and the specificity was 47.62% and 100%, respectively. FIG. 9 is a ROC curve using serum TIMP-1 level as a diagnostic marker for epileptic and normal controls, where TIMP-1 concentration was 74.35ng/ml, the sensitivity of TIMP-1 as a diagnostic indicator for epilepsy and normal controls was 66.52%, the specificity was 59.80%, and AUC was 0.594.
While the present invention has been described in detail with reference to the embodiments, it should not be construed as limited to the scope of the patent. Various modifications and changes may be made by those skilled in the art without inventive step within the scope of the appended claims.

Claims (4)

1. A liquid chip kit for detecting intractable epilepsy is characterized in that: comprises coating microspheres, a biotin-labeled detection antibody, streptomycin-R phycoerythrin, a reaction buffer solution and a reaction diluent; wherein,
the coated microspheres comprise microspheres coated with a TIMP-1 capture antibody, microspheres coated with an MMP2 capture antibody, microspheres coated with an MMP3 capture antibody and microspheres coated with an MMP9 capture antibody, wherein the concentration of each microsphere is 3 multiplied by 106～8×106Per mL;
the detection antibody comprises a TIMP-1 detection antibody, an MMP2 detection antibody, an MMP3 detection antibody and an MMP9 detection antibody, and the concentration of each detection antibody is 0.08-0.12 mu g/mL;
the reaction buffer solution is a PBS buffer solution with the pH value of 7.0-7.5;
the dilution buffer is PBS buffer containing 0.05 wt% -0.15 wt% BSA, and the pH value is 7.0-7.5.
2. The liquid phase chip kit according to claim 1, wherein: the concentration of each coating microsphere in the kit is 5 multiplied by 106one/mL.
3. The liquid phase chip kit according to claim 1, wherein: the concentration of each detection antibody in the kit is 0.1 mug/mL.
4. The liquid phase chip kit according to claim 1, wherein: the mass fraction of BSA in the dilution buffer was 0.1 wt%.
CN201910120115.7A 2019-02-18 2019-02-18 A kind of liquid phase chip reagent box detecting intractable epilepsy disease Pending CN109682969A (en)

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Publication number Priority date Publication date Assignee Title
CN112285346A (en) * 2020-07-25 2021-01-29 四川大学华西医院 High-sensitivity HIV p24 detection method based on quantum dot selective cation exchange reaction
CN112285346B (en) * 2020-07-25 2024-02-13 四川大学华西医院 High-sensitivity HIV p24 detection method based on quantum dot selective cation exchange reaction

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Application publication date: 20190426