CN111044719A - Electrochemiluminescence kit for detecting titin antibody and preparation method thereof - Google Patents
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Abstract
The invention relates to an electrochemiluminescence kit for detecting a Titin antibody (Titin antibody) from blood and a preparation method thereof, wherein the prepared kit comprises the following components: the kit comprises streptavidin coupled magnetic particle working solution, biotin labeled Titin antigen working solution, terpyridyl ruthenium labeled anti-human IgG antibody working solution, Titin antibody calibrator and/or quality control working solution, tripropylamine-containing electrochemiluminescence substrate solution, cleaning solution, a luminophore system of the kit is electrochemiluminescence, a streptavidin-biotin signal amplification system is utilized, the detection sensitivity is high, the linear range is wide, the detection result repeatability is good, and the accurate quantification of the Titin antibody can be realized.
Description
Technical Field
The invention relates to an electrochemiluminescence kit for detecting a Titin antibody (Titin antibody) and a preparation 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 the control is not timely and effective, 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. 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 the postsynaptic membrane of NMJ, and acetylcholine receptor antibody (AChRAb) is combined with acetylcholine receptor (AChR) on NMJ, resulting in decreased AChR number and loss of function, and is the main immunological pathogenic mechanism of AChRAb positive MG, and the positive rate of the acetylcholine receptor antibody (AChR antibody) in MG is 85% -90%. However, AchR antibody is not present in all MG patients and is not associated with disease severity, whether thymoma is complicated, and age of onset. In recent years, it has been found that antibodies against skeletal muscle and myocardial striated muscle components, including myosin, actin, filamin, etc., are present in the serum of some MG patients and these antibodies are called striated muscle antibodies. There are 2 striated muscle antibodies, myoglobin (tin) and Ryanodine receptor (RYR) antibodies, in the sera of about 95% of patients with thymoma and 50% of patients with late-onset MG[2]。
Where tins, also known as Titin or connexin, are the third structural protein present in skeletal and cardiac muscle, in addition to the thick and thin fibers. It is a larger protein of all known proteins, has the relative molecular mass of 300 KDa-3000 KDa, and is important for maintaining the elasticity of cells and the contraction movement of muscles[3]. Most Titin antibodies recognize a specific region of the Titin protein with a relative molecular mass of about 30kDa, called myseteria gravis tin-30(MGT-30), which is the major immunogenic region of Titin[4]. Titin is localized in muscle cells, and Titin antibodies do not generally cause muscle weakness symptoms per se. Titin antibodies have good sensitivity and specificity for diagnosis of thymoma combined with MG (MGT). Foreign studies show that the positive rate of Titin antibody in the serum of MG patients is 20-40%, the positive rate of Titin antibody in MGT patients is 49-95%, and the positive rate of Titin antibody in the serum of MGT patients is at age>The positive rate of the Titin antibody in 60-year-old delayed MG patients is 60-80%[5]. The current method for detecting Titin antibody is enzyme linked immunosorbent assay (ELISA), and there are commercial ELISA kits for detecting Titin antibody. However, the inherent sensitivity of the ELISA method is low, the result repeatability is low, the operation is complicated, and the like, so that the Titin antibody can not be accurately and simply determined.
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 quickly, low-risk and accurately and quantitatively detecting the Titin antibody does not exist in the prior art, an electrochemiluminescence kit for detecting the Titin antibody and a preparation method thereof are provided.
The technical scheme adopted by the invention for solving the technical problems is as follows:
an electrochemiluminescence kit for detecting a Titin antibody, comprising: streptavidin coupled magnetic particle working solution, biotin labeled Titin antigen working solution, terpyridyl ruthenium labeled anti-human IgG antibody working solution, tripropylamine-containing electrochemiluminescence substrate solution and cleaning solution.
Preferably, the Titin antigen is an externally purchased MGT-30 recombinant protein. The main immunogen region MGT-30 protein of the titin is recombined and expressed, 3 sections of main fragments IG-IF-FN (IG 108-110) are cloned, and the purified MGT-30 recombinant protein is obtained through a prokaryotic expression system.
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 Titin 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 Titin antibody further comprises a Titin antibody calibrator working solution and/or a quality control working solution, wherein the Titin antibody calibrator working solution and/or the quality control working solution is prepared from one of phosphate buffer solution, Tris buffer solution, HEPES buffer solution and MES buffer solution, the pH 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 Titin antibody, which comprises the following steps: the method comprises the steps of preparing a streptavidin coupled magnetic particle working solution, preparing a biotin labeled Titin 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 Titin antigen comprises the following steps: mixing biotin activated by N-hydroxysuccinimide and Titin antigen uniformly 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 Titin antigen marked by the biotin.
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 Titin 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 Titin 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 the Titin antibody in the sample can be quantitatively detected only by arranging a standard curve in the test software, so that the detection is quicker, safer, 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 Titin 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 Titin antigen
Taking 1mg of recombinant Titin 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 Titin 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 NHS-Biotin and the antigen according to a molar ratio of 5:1 at room temperature for 1 hour to obtain a Biotin-labeled Titin antigen solution;
transferring the biotin-labeled Titin 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 Titin antigen after dialysis, putting the Titin antigen into a centrifuge tube or a freezing storage tube, and freezing and storing the Titin antigen for later use at the temperature of less than or equal to-20 ℃.
(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) Preparing a biotin-labeled Titin antigen working solution and a terpyridyl ruthenium-labeled anti-human IgG antibody working solution to prepare a phosphate buffer solution with the pH of 6.5 and the 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 TritonX-100 and 0.2 wt% of proclin 300; then, biotin-labeled Titin antigen working solution with the antibody concentration of 0.8mg/L and terpyridyl ruthenium-labeled anti-human IgG antibody working solution with the antibody concentration of 8mg/L are prepared by using the buffer solution.
(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 preparing a Titin antibody calibrator and a quality control working solution by using the buffer solution, wherein the calibrator has 5 points, and the concentrations of the Titin antibody are respectively 80pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL and 3000 pg/mL. The quality control product has two concentrations, namely high concentration and low concentration, and the concentration of the Titin antibody is 100pg/mL and 3500pg/mL respectively.
(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 with pH of 6.5, 0.05 wt% of dodecanol glycol ether and 0.01 wt% of proclin300 are prepared.
(8) Subpackaging and assembling a reagent, namely subpackaging 12 ml/bottle of streptavidin coupled magnetic particle working solution, 12 ml/bottle of biotin labeled Titin 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 at 20-25 ℃.
Embodiment 2 the method for detecting a Titin antibody by using the kit of the invention takes a full-automatic electrochemiluminescence immunoassay analyzer as a detection instrument, and the kit is loaded on the instrument for detection, and the steps are as follows:
adding a sample (or a calibrator or a quality control material), a biotin-labeled Titin 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. Calculating the content of Titin antibody 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 80pg/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 41.5pg/mL according to the above method, and the sensitivity of the reagent was determined to be 60pg/mL or less based on the results.
TABLE 1 results of sensitivity measurement of the kit of the present invention
Example 4 study of reproducibility of the kit of the invention
The detection concentration of samples with the concentrations of 125pg/mL and 3765pg/mL is 10 times, the Coefficient of Variation (CV) of each sample is calculated respectively, and the result shows that the CV of the kit is less than 4%.
TABLE 2 repeatability measurements of the kits of the invention
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 Titin antibody standard substances with the concentration of 95, 1265 and 3495pg/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 substance of the kit is less than 10%.
TABLE 3 accuracy determination results of the kit of the present invention
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]Gilhus,N.E.,Skeie,G.O.,Romi,F.,Lazaridis,K.,Zisimopoulou,P.&Tzartos,S.(2016) Myasthenia gravis:autoantibody characteristics and theirimplications for therapy.Nat Rev Neurol. 12(5):259-268.
[3]Powers,K.,Schappacher-Tilp,G.,Jinha,A.,Leonard,T.,Nishikawa,K.&Herzog,W.(2014) Titin force is enhanced in actively stretched skeletalmuscle.J Exp Biol.217(20),3629-3636.
[4]Romi,F.,Skeie,G.O.,Gilhus,N.E.&Aarli,J.A.(2005)Striationalantibodies in myasthenia gravis:reactivity and possible clinical signifificance.Arch Neurol.62(3),442-446.
[5]Suzuki,S.,Utsugisawa,K.,Nagane,Y.&Suzuki,N.(2011)Three types ofstriational antibodies in myasthenia gravis.Autoimmune Dis.2011,740583。
Claims (10)
1. an electrochemiluminescence kit for detecting a Titin antibody (Titin antibody), comprising: streptavidin coupled magnetic particle working solution, biotin labeled Titin 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 the Titin antibody according to claim 1, wherein the Titin 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 a Titin 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, and 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 Titin antibody according to any one of claims 1 to 3, wherein the working solution of biotin-labeled Titin antigen and terpyridyl ruthenium-labeled anti-human IgG antibody is prepared from one of phosphate buffer solution, Tris buffer solution, HEPES buffer solution and MOPSO buffer solution with pH of 6.0-7.6 and 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 dodecyl polyglycol ether and/or polyethylene glycol p-isooctyl phenyl ether and/or polyoxyethylene sorbitan monolaurate and/or polyoxyethylene lauryl alcohol, 0.05-0.5 wt% proclin300 and/or sodium azide.
5. The electrochemiluminescence kit for detecting Titin antibody according to any one of claims 1 to 4, it is characterized in that the electrochemical luminescence kit for detecting the Titin antibody also comprises Titin antibody calibrator working solution and/or quality control material working solution, the Titin 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 a Titin antibody according to any one of claims 1 to 5, wherein the cleaning solution is KOH having a pH of 13 or more and a concentration of 0.1mol/L to 0.5mol/L, and the cleaning solution contains 0.01 to 2 wt% of an alkyl polyethylene glycol surfactant.
7. The electrochemiluminescence kit for detecting Titin antibody of 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% of alkyl polyethylene glycol surfactant, 0.05-0.5 wt% of proclin300 and/or sodium azide.
8. A method for preparing an electrochemiluminescence kit for detecting a Titin 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 Titin 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 Titin antibody according to claim 8, wherein the method for preparing the biotin-labeled Titin antigen comprises the following steps: uniformly mixing biotin activated by N-hydroxysuccinimide and Titin 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 Titin antigen marked by the biotin.
10. The method for preparing the electrochemiluminescence kit for detecting the Titin antibody according to claim 8 or 9, wherein the method for preparing 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.
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