CN110988336A - Electrochemiluminescence kit for detecting muscle-specific tyrosine kinase (MuSK) antibody and preparation method - Google Patents

Electrochemiluminescence kit for detecting muscle-specific tyrosine kinase (MuSK) antibody and preparation method Download PDF

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CN110988336A
CN110988336A CN201911255089.5A CN201911255089A CN110988336A CN 110988336 A CN110988336 A CN 110988336A CN 201911255089 A CN201911255089 A CN 201911255089A CN 110988336 A CN110988336 A CN 110988336A
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孟君
泮锋纲
丁俊杰
施启尧
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Sunlant Biological Engineering Co ltd
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Abstract

The invention relates to an electrochemiluminescence kit for detecting muscle specific tyrosine kinase (MuSK) antibody from blood and a preparation method thereof, and the prepared kit comprises: the kit comprises streptavidin coupled magnetic particle working solution, biotin labeled MuSK antigen working solution, terpyridyl ruthenium labeled anti-human IgG antibody working solution, MuSK antibody calibrator and/or quality control product working solution, tripropylamine-containing electrochemiluminescence substrate solution, cleaning solution, and a luminophore system of the kit is electrochemiluminescence.

Description

Electrochemiluminescence kit for detecting muscle-specific tyrosine kinase (MuSK) antibody and preparation method
Technical Field
The invention relates to an electrochemiluminescence kit for detecting a MuSK antibody and a use method thereof, belonging to the technical field of immunoassay.
Background
Autoimmune diseases are diseases caused by the immune system generating immune reaction to the components of the body, causing damage. Normally, the immune system reacts only to foreign bodies, such as bacteria, viruses, parasites, and transplants, which invade the body, and destroy or repel the foreign bodies. Under the influence of some factors, some abnormalities occur in the tissue components of the body or the immune system itself, so that the immune system can attack the self components as foreign matters by mistake. At this time, the immune system produces antibodies and active lymphocytes against some components of the body itself, and damages and destroys the organs of the body, resulting in diseases. If not timely and effectively controlled, the fruit is serious: attacks of the immune system affect every human organ and usually attack it for life; it is also sometimes possible to cause damage to numerous parts of the body at the same time. Early diagnosis is important for managing self-avoidance, where early detection can avoid or delay the occurrence of irreparable damage to the target organ or tissue.
Myasthenia Gravis (MG) is an acquired autoimmune disease in which autoantibodies cause, cellular immune-dependent, complement involvement mainly involve the postsynaptic membrane of the neuromuscular junction, resulting in transmission failure of the neuromuscular junction and skeletal muscle contraction weakness. The main clinical manifestations of the medicine are myasthenia of skeletal muscles, easy fatigue, aggravation after activity, and obvious alleviation and relief of symptoms after rest and application of cholinesterase inhibitor. It is characterized by wide disease age, high disability rate, easy recurrence and poor prognosis, and seriously threatens human health. The global prevalence rate is 15/100-300/100 thousands, and the annual incidence rate is 10/100 thousands[1]. MG can occur at all ages. Prior to age 40, women have a higher incidence than men; the incidence rate of the male and female is equivalent to 40-50 years old; after age 50, men have a slightly higher incidence than women.
MG is a typical antibody-mediated autoimmune disease, the main target antigen is acetylcholine receptor (AChR) of postsynaptic membrane of NMJ, the acetylcholine receptor antibody is combined with acetylcholine receptor (AChR) on NMJ, which results in decrease of AChR quantity and loss of function, the main immunological pathogenic mechanism of AChRAb positive MG, and the positive rate of the acetylcholine receptor antibody (AchR-Ab) in MG is 85% -90%. Acetylcholine receptor antibody (AchR-Ab) positive in the serum of MG patients is called seropositive MG (SPMG), but in the research, 10% -20% of MG patients are found to be seronegative for AChRAb, and called seronegative for myasthenia gravis (SNMG), which is the important research point of the pathogenesis of MG in recent years. In recent years, the research finds that muscle-specific tyrosine kinase antibody (MuSKAb) exists in the serum of SNMG patients, and the clinical characteristics of the MuSKAb positive SNMG patients are similar to AClear distinction among ChRAb positive patients[2]。
The tyrosine kinase receptor (MuSK) is a transmembrane polypeptide with the molecular weight of 110kD, is selectively expressed on skeletal muscle and electric organ of Torpedo, the extracellular region of the receptor is composed of 4 IgG-like regions and 1 cysteine aggregation region, and the intracellular region is composed of 1 membrane-proximal region, 1 kinase region and a carboxyl terminal tail. MuSK is a component of a receptor of neurogenic proteins essential for the development of neuromuscular junctions, which plays an important role in the formation of neuromuscular synapses[3]. MuSK antibody can be detected in the blood of part of SNMG patients, and the positive rate of the MuSK antibody is 1-10%[4]. Unlike AChR antibodies, the MuSK antibody class mostly belongs to the IgG4 subtype. MuSK does not cause activation of complement at end plate, but directly plays a pathogenic role, reduces the density of post-synaptic membrane AChR, destroys the structure of NMJ[5]. Fluctuations in MuSK antibody titers in individual patients are correlated with the severity of the disease, so changes in antibody titers can reflect the activity of the disease. Has obvious female susceptibility tendency, rapid deterioration in early disease course, and easy occurrence of crisis, and is rare in Chinese. The current MuSK antibody detection method mainly comprises the following steps: RIPA (radio-immune assay), ELISA (enzyme-linked immunosorbent assay) detection, wherein the RIPA method has radioactive risk, short reagent expiration date, inconvenient use and substantial elimination. Although the ELISA method overcomes the radioactivity risk, the accurate and simple MuSK antibody determination is difficult to realize due to the inherent defects of low sensitivity, low result repeatability, complex operation and the like of the ELISA method.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: in order to solve the technical problem that a method or a kit for rapidly and accurately quantitatively detecting the MuSK antibody does not exist in the prior art, an electrochemiluminescence kit for detecting the MuSK antibody and a preparation method are provided.
The technical scheme adopted by the invention for solving the technical problems is as follows:
an electrochemiluminescence kit for detecting a MuSK antibody, comprising: the kit comprises streptavidin coupled magnetic particle working solution, biotin labeled MuSK antigen working solution, terpyridyl ruthenium labeled anti-human IgG antibody working solution, tripropylamine-containing electrochemiluminescence substrate solution and cleaning solution.
The antigen is transfected into escherichia coli by an expression vector containing a coding human recombinant MuSK polypeptide (hMuSK) by using a biological engineering technology, the expression is induced by isopropyl thio- β -galactoside (IPTG), and the SDS-PAGE electrophoresis of a thallus lysate confirms.
Preferably, the anti-human IgG antibody is an externally purchased monoclonal antibody, polyclonal antibody, monoclonal antibody Fab fragment or polyclonal antibody Fab fragment, preferably from mouse, rabbit, sheep.
Preferably, the streptavidin coupled magnetic particle working solution is prepared from one of phosphate buffer solution, Tris buffer solution, HEPES buffer solution and MOPSO buffer solution with the pH of 6.8-7.6 and the concentration of 0.01-0.2 mol/L, wherein the buffer solution contains 0.05-2 wt% of bovine serum albumin and/or casein, 0.02-1 wt% of dodecyl polyethylene glycol ether (Brij35) and/or polyethylene glycol p-isooctyl phenyl ether (TritonX-100) and/or polyoxyethylene sorbitan monolaurate (Tween20) and/or polyoxyethylene lauryl ether (peregal O-20), 0.05-0.5 wt% of proclin300 and/or sodium azide.
Preferably, the biotin-labeled MuSK antigen and terpyridyl ruthenium-labeled anti-human IgG antibody working solution is prepared from one of phosphate buffer solution, Tris buffer solution, HEPES buffer solution and MOPSO buffer solution with the pH of 6.0-7.6 and the concentration of 0.01-0.2 mol/L, wherein the buffer solution contains 0.5-5 wt% of bovine serum albumin, 1-100 mg/L of anti-interference agent A, 0.5-5 wt% of sodium chloride, 1-5 wt% of sucrose, 0.05-2 wt% of calf serum, 0.02-1 wt% of casein, 0.02-1 wt% of Brij35 and/or TritonX-100 and/or Tween20 and/or peregal O-20, and 0.05-0.5 wt% of proclin300 and/or sodium azide.
Preferably, the electrochemiluminescence kit for detecting the MuSK antibody further comprises a MuSK antibody calibrator working solution and/or a quality control working solution, wherein the MuSK antibody calibrator working solution and/or the quality control working solution is prepared from one of a phosphate buffer solution, a Tris buffer solution, a HEPES buffer solution and a MES buffer solution, the pH value of the phosphate buffer solution is 6.0-7.6, the concentration of the phosphate buffer solution is 0.01-0.2 mol/L, the buffer solution contains 0.5-5 wt% of bovine serum albumin, 10-50 wt% of human serum, 0.5-3 wt% of sodium chloride, 2-20 wt% of ethylene glycol, 0.02-1 wt% of Brij35 and/or Triton X-100 and/or Tween20 and/or peregal O-20, and 0.05-0.5 wt% of proclin300 and/or sodium azide.
Preferably, the cleaning solution is KOH solution with pH of more than 13 and concentration of 0.1 mol/L-0.5 mol/L, and the cleaning solution contains 0.01-2 wt% of alkyl polyethylene glycol surfactant.
Preferably, the pH of the chemiluminescent substrate solution is 6.0-7.2, the concentration of the chemiluminescent substrate solution is 0.05-0.4 mol/L phosphate buffer solution or Tris buffer solution, and the buffer solution contains 0.05-0.4 mol/L tripropylamine, 0.01-2 wt% of alkyl polyethylene glycol surfactant, 0.05-0.5 wt% of proclin300 and/or sodium azide.
The invention also provides a preparation method of the electrochemiluminescence kit for detecting the MuSK antibody, which comprises the following steps: the method comprises the steps of preparing a streptavidin coupled magnetic particle working solution, preparing a biotin labeled MuSK antigen working solution, preparing a terpyridyl ruthenium labeled anti-human IgG antibody working solution, preparing a cleaning solution, preparing an electrochemiluminescence substrate solution, and subpackaging and assembling the prepared reagents.
Preferably, the preparation method of the biotin-labeled MuSK antigen comprises the following steps: and uniformly mixing biotin activated by N-hydroxysuccinimide and MuSK antigen according to the molar ratio of 1-20:1 at the temperature of 2-8 ℃ or room temperature for 0.5-2 hours, and dialyzing to remove redundant biotin to obtain the biotin-labeled MuSK antigen.
Preferably, the preparation method of the ruthenium terpyridyl labeled anti-human IgG antibody comprises the following steps: mixing an anti-human IgG antibody solution with the pH value of 6.5-8.0 with a Ru-NHS ester solution to ensure that the mass ratio of the anti-human IgG antibody to the Ru-NHS ester is 5-20:1, carrying out a light-shielding reaction at 37 ℃ for 1-3 hours, then adding a glycine solution to terminate the reaction, and dialyzing the reaction solution to remove redundant Ru-NHS ester to obtain the terpyridyl ruthenium labeled anti-human IgG antibody.
The invention has the beneficial effects that:
the invention provides an electrochemiluminescence kit for detecting a MuSK antibody and a preparation method, wherein a luminous system of the prepared kit is electrochemiluminescence, a streptavidin-biotin signal amplification system is utilized, the detection sensitivity is high, the linear range is wide, the repeatability of a detection result is good, and the accurate quantification of the MuSK antibody can be realized; particularly, the kit is used for a full-automatic electrochemical luminescence system, the steps of sample adding, incubation, cleaning, detection and the like can be automated, the result deviation caused by manual operation is avoided, the working efficiency is improved, and only a test sample needs to be arranged in the test software, so that the MuSK antibody in the sample can be quantitatively detected, and the detection is quicker, more reliable and more stable.
Detailed Description
The present invention will now be described in further detail.
Example 1 preparation method of electrochemiluminescence kit for detecting MuSK antibody
(1) Preparation of streptavidin coupled magnetic particle working solution
The streptavidin-coupled magnetic particle suspension was magnetically separated from the supernatant, resuspended at a concentration of 0.5mg/mL using 0.1mol/L PBS buffer containing 0.5 wt% bovine serum albumin, 0.05 wt% Triton X-100, 0.05 wt% proclin300, and 0.05 wt% sodium azide at pH 6.8.
(2) Preparation of Biotin-labeled MuSK antigen
Taking 1mg of recombinant MuSK antigen, adding a proper amount of PBS (0.05M, pH7.0-7.6) to adjust the total concentration of the antibody to 1mg/ml, adding the recombinant MuSK antigen into a dialysis bag for dialysis, changing the dialysate every 3-4 hours for 3-4 times, and transferring the antigen into a centrifuge tube or a freezing storage tube after dialysis;
accurately weighing 1mg of N-hydroxysuccinimide activated Biotin (NHS-Biotin), adding a proper amount of water to adjust the concentration of the Biotin to 2mg/ml, and obtaining an NHS-Biotin aqueous solution;
adding an NHS-Biotin aqueous solution into an antigen solution, and uniformly mixing the NHS-Biotin and the antigen according to a molar ratio of 5:1 at room temperature for 1 hour to obtain a Biotin-labeled MuSK antigen solution; transferring the biotin-labeled MuSK antigen solution to a dialysis bag for dialysis (the dialysate is 0.1M PBS, pH7.4), changing the dialysate every 3-4 hours for 3-4 times, collecting the biotin-labeled MuSK antigen after dialysis, putting the MuSK antigen into a centrifuge tube or a freezing storage tube, and freezing and storing the MuSK antigen for later use at the temperature of-20 ℃ or below.
(3) Preparation of ruthenium terpyridyl-labeled anti-human IgG antibody
Preparing a Ru-NHS ester solution with the concentration of 10mg/mL by using DMSO; preparing an anti-human IgG antibody solution with the concentration of 1mg/mL by using 0.1M phosphate buffer solution with the pH value of 7.4;
adding 10 mu L of prepared Ru-NHS ester solution into 1mL of anti-human IgG antibody solution, reacting for 2 hours at 37 ℃ in a dark place, then adding 20 mu L of 2M glycine to stop the reaction, dialyzing the reaction solution in 0.1M PBS buffer solution with pH7.4 overnight, changing the solution for 3 times in the process, recovering the terpyridyl ruthenium-labeled anti-human IgG antibody solution, and preserving for later use at the temperature of less than or equal to-20 ℃ in a freezing way.
(4) Preparation of biotin-labeled MuSK antigen working solution and terpyridyl ruthenium-labeled anti-human IgG antibody working solution
Preparing a phosphate buffer solution with pH of 6.5 and concentration of 0.1mol/L, wherein the buffer solution contains 2 wt% of bovine serum albumin, 0.1 wt% of casein, 1 wt% of sodium chloride, 1 wt% of sucrose, 2 wt% of calf serum, 0.5 wt% of Triton X-100 and 0.2 wt% of proclin 300; then, the buffer solution is used for preparing a biotin-labeled MuSK antigen working solution with the antibody concentration of 1mg/L and an anti-human IgG antibody working solution with the antibody concentration of 10mg/L and terpyridyl ruthenium-labeled.
(5) Preparation of working solution of calibrator and quality control material
Preparing a 0.1mol/L N- (2-hydroxyethyl) piperazine-N' (2-ethanesulfonic acid) (HEPES) buffer solution with a pH of 7.4, wherein the buffer solution contains 1 wt% of bovine serum albumin, 20 wt% of human serum, 1 wt% of sodium chloride, 5 wt% of ethylene glycol, 0.1 wt% of Tween-20 and 0.2 wt% of proclin 300; then, a MuSK antibody calibrator and a quality control working solution are prepared by using the buffer solution, wherein the concentrations of the MuSK antibody are respectively 40pg/mL, 100pg/mL, 250pg/mL, 500pg/mL and 2000pg/mL at 5 points in total. The quality control product has two concentrations, namely high concentration and low concentration, and the concentrations of the MuSK antibody are respectively 50pg/mL and 900 pg/mL.
(6) Preparation of cleaning solution
Preparing 176mmol/L KOH solution, and the prepared cleaning solution contains 0.15% of lauryl alcohol glycol ether.
(7) Preparation of chemiluminescent substrate solution
0.15mol/L tripropylamine solution with pH of 6.5 is prepared, which contains 0.05 wt% of lauryl alcohol glycol ether and 0.01 wt% of proclin 300.
(8) Subpackaging and assembling a reagent, namely subpackaging 12 ml/bottle of streptavidin-coupled magnetic particle working solution, 12 ml/bottle of biotin-labeled MuSK antigen working solution, 12 ml/bottle of terpyridyl ruthenium-labeled anti-human IgG antibody working solution and 1.0 ml/bottle of calibrator/quality control working solution, assembling the components together, storing the components at 2-8 ℃, and individually packaging 380 ml/bottle of cleaning solution and 380 ml/bottle of chemiluminescent substrate solution and storing the components at 20-25 ℃.
Embodiment 2 a method for detecting a MuSK antibody using the kit of the present invention uses a full-automatic electrochemiluminescence immunoassay analyzer as a detection instrument, and the kit is loaded on the instrument for detection, comprising the following steps:
adding a sample (or a calibrator or a quality control material), a biotin-labeled MuSK antigen working solution and a terpyridyl ruthenium-labeled anti-human IgG antibody working solution into a reaction cup, and incubating for 10 minutes at 37 ℃ to form an antigen-antibody-anti-antibody complex solution;
adding streptavidin-coated magnetic particle working solution into the antigen-antibody-anti-antibody compound solution, and incubating for 10 minutes at 37 ℃ to form magnetic compound suspension;
placing the magnetic compound suspension in a magnetic field, flowing a cleaning solution through the magnetic field, and washing the magnetic compound;
and injecting electrochemiluminescence substrate liquid into the washed magnetic compound in sequence, and detecting the photon intensity of the electrochemiluminescence substrate liquid. The content of the MuSK antibody is calculated according to the photon intensity and the calibration curve.
Performance evaluation of the kit of the present invention
EXAMPLE 3 investigation of reagent sensitivity
Reagent sensitivity was determined based on the lowest limit of detection (LOB) which was performed as described below. Detecting the zero concentration calibrator 20 times to obtain a signal value (RLU) of 20 measurement results, calculating the average value M and standard deviation SD to obtain an RLU value corresponding to M +2SD, performing two-point regression fitting according to the concentration-RLU value result between the zero concentration calibrator and an adjacent concentration calibrator (the adjacent concentration is 40pg/mL) to obtain a linear equation, substituting the RLU value corresponding to M +2SD into the equation, and calculating to obtain a corresponding concentration, namely a lowest detection Limit (LOB).
The results of the following Table 1 show that the lowest limit of detection (LOB) of the reagent was 29.3pg/mL according to the above method, and the sensitivity of the reagent was determined to be 30pg/mL or less based on the results.
TABLE 1 results of sensitivity measurement of the kit of the present invention
Figure BDA0002310020290000041
Example 4 study of reproducibility of the kit of the invention
The detection concentration is 45.6pg/mL and 987pg/mL, each sample is detected for 10 times, the Coefficient of Variation (CV) of each sample is calculated respectively, and the result shows that the CV of the coefficient of variation of the kit is less than 4%.
TABLE 2 repeatability measurements of the kits of the invention
Figure BDA0002310020290000051
Example 5 accuracy measurement results of the kit of the present invention
As the index does not have national or international standard products at present, the accuracy of the kit is measured by adopting a third-party outsourcing standard product. The MuSK antibody standard samples with the concentration of 65, 256 and 1100pg/mL are detected, the deviation of the detection value and the theoretical value is calculated respectively, and the result shows that the deviation of the detection standard sample of the kit is less than 10%.
TABLE 3 accuracy determination results of the kit of the present invention
Figure BDA0002310020290000052
It is to be understood that the invention disclosed is not limited to the particular methodology, protocols and materials described, as such is intended to limit the scope of the invention, which is limited only by the claims appended hereto.
Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
Reference documents:
[1]Carr,A.S.,Cardwell,C.R.,McCarron,P.O.&McConville,J.(2010)Asystematic review of population based epidemiological studies in MyastheniaGravis.BMC Neurol.10(1),46
[2]Bartoccioni,E.,Marino,M.,Evoli,A.,Ruegg,M.A.,Scuderi,F.&Provenzano,C.(2003)Identification of disease-specific autoantibodies inseronegative myasthenia gravis.Ann N Y Acad Sci.998,356-358.
[3]Liyanage,Y.,Hoch,W.,Beeson,D.&Vincent,A.(2002)The Agrin/muscle-specific kinase pathway:new targets for autoimmune and genetic disorders atthe neuromuscular junction.Muscle Nerve.25(1),4-16.
[4]Querol,L.&Illa,I.(2013)Myasthenia gravis and the neuromuscularjunction.Curr Opin Neurol.26(5),459-465.
[5]Plomp,J.J,Huijbers,M.G.,van der Maarel,S.M.&Verschuuren,J.J.(2012)Pathogenic IgG4 subclass autoantibodies in MuSK myasthenia gravis.Ann N YAcad Sci.1275,114-122.

Claims (10)

1. an electrochemiluminescence kit for detecting MuSK antibodies, comprising: the kit comprises streptavidin coupled magnetic particle working solution, biotin labeled MuSK antigen working solution, terpyridyl ruthenium labeled anti-human IgG antibody working solution, tripropylamine-containing electrochemiluminescence substrate solution and cleaning solution.
2. The electrochemiluminescence kit for detecting a MuSK antibody according to claim 1, wherein the MuSK antibody is a monoclonal antibody, a polyclonal antibody, a monoclonal antibody Fab fragment, or a polyclonal antibody Fab fragment.
3. The electrochemiluminescence kit for detecting MuSK antibody according to claim 1 or 2, wherein the streptavidin-coupled magnetic particle working solution is prepared from one of phosphate buffer, Tris buffer, HEPES buffer, and MOPSO buffer with pH of 6.8-7.6 and concentration of 0.01-0.2 mol/L, wherein the buffer contains 0.05-2 wt% of bovine serum albumin and/or casein, 0.02-1 wt% of dodecyl polyethylene glycol ether and/or polyethylene glycol p-isooctyl phenyl ether and/or polyoxyethylene sorbitan monolaurate and/or polyoxyethylene lauryl ether, and 0.05-0.5 wt% of proclin300 and/or sodium azide.
4. The electrochemiluminescence kit for detecting MuSK antibody according to any one of claims 1-3, wherein the working solution of biotin-labeled MuSK antigen and terpyridyl ruthenium-labeled anti-human IgG antibody is prepared from one of phosphate buffer, Tris buffer, HEPES buffer, and MOPSO buffer with pH of 6.0-7.6 and concentration of 0.01-0.2 mol/L, wherein the buffer contains 0.5-5 wt% of bovine serum albumin, 1-100 mg/L of anti-interference agent A, 0.5-5 wt% of sodium chloride, 1-5 wt% of sucrose, 0.05-2 wt% of calf serum, 0.02-1 wt% of casein, 0.02-1 wt% of dodecyl poly (vinyl alcohol) and/or polyethylene glycol p-isooctyl phenyl ether and/or polyoxyethylene sorbitan monolaurate and/or polyoxyethylene lauryl ether, and/or polyoxyethylene lauryl ether, 0.05-0.5 wt% proclin300 and/or sodium azide.
5. The electrochemiluminescence kit for detecting MuSK antibodies according to any one of claims 1-4, it is characterized in that the electrochemiluminescence kit for detecting the MuSK antibody also comprises MuSK antibody calibrator working solution and/or quality control material working solution, the MuSK antibody calibrator and/or quality control working solution is prepared from one of phosphate buffer solution, Tris buffer solution, HEPES buffer solution and MES buffer solution with the pH of 6.0-7.6 and the concentration of 0.01-0.2 mol/L, the buffer solution contains 0.5-5 wt% of bovine serum albumin, 10-50 wt% of human serum, 0.5-3 wt% of sodium chloride, 2-20 wt% of ethylene glycol, 0.02-1 wt% of Brij35 and/or Triton X-100 and/or Tween20 and/or peregal O-20, and 0.05-0.5 wt% of proclin300 and/or sodium azide.
6. The electrochemiluminescence kit for detecting MuSK antibody according to any of claims 1-5, wherein the cleaning solution is KOH with a pH of 13 or higher and a concentration of 0.1mol/L to 0.5mol/L, and the cleaning solution contains 0.01 to 2 wt% of alkyl polyethylene glycol surfactant.
7. The kit for detecting MUSK antibodies as claimed in any one of claims 1 to 6, wherein the electrochemiluminescence substrate solution is phosphate buffer or Tris buffer with pH of 6.0-7.2 and concentration of 0.05-0.4 mol/L, and the buffer comprises 0.05-0.4 mol/L tripropylamine, 0.01-2 wt% alkylpolyethylene glycol surfactant, 0.05-0.5 wt% proclin300 and/or sodium azide.
8. A method for preparing an electrochemiluminescence kit for detecting a MuSK antibody is characterized by comprising the following steps: the method comprises the steps of preparing a streptavidin coupled magnetic particle working solution, preparing a biotin labeled MuSK antigen working solution, preparing a terpyridyl ruthenium labeled anti-human IgG antibody working solution, preparing a cleaning solution, preparing an electrochemiluminescence substrate solution, and subpackaging and assembling the prepared reagents.
9. The method for preparing the electrochemiluminescence kit for detecting the MuSK antibody according to claim 8, wherein the method for preparing the biotin-labeled MuSK antigen comprises the following steps: uniformly mixing biotin activated by N-hydroxysuccinimide and MuSK antigen according to the molar ratio of 1-20:1 at the temperature of 2-8 ℃ or room temperature for 0.5-2 hours, and dialyzing or passing through a column to remove redundant biotin to obtain the biotin-labeled MuSK antigen.
10. The method for preparing the electrochemiluminescence kit for detecting the MuSK antibody according to claim 8 or 9, wherein the ruthenium terpyridyl label anti-human IgG antibody is prepared by the following steps: mixing an anti-human IgG antibody solution with the pH value of 6.5-8.0 with a Ru-NHS ester solution to ensure that the mass ratio of the anti-human IgG antibody to the Ru-NHS ester is 5-20:1, carrying out a light-shielding reaction at 37 ℃ for 1-3 hours, then adding a glycine solution to terminate the reaction, and dialyzing the reaction solution to remove redundant Ru-NHS ester to obtain the terpyridyl ruthenium labeled anti-human IgG antibody.
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