阅读说明：本技术 空间邻近化学发光法检测血清淀粉样蛋白a试剂盒的应用及检测方法 (Application of kit for detecting serum amyloid A by using space proximity chemiluminescence method and detection method ) 是由 奚伟红 史兵伟 廖鸳鸯 朱丹丹 于 2018-09-25 设计创作，主要内容包括：本发明涉及一种空间邻近化学发光法检测血清淀粉样蛋白A的试剂盒及其检测方法。试剂盒包括：酶标记物、发光标记物、辅助剂、触发剂和校准品；其中,校准品包括不同浓度SAA抗原的校准品和0.1M磷酸盐稀释液；酶标记物包括过氧化物酶标记的SAA检测抗体和0.05M磷酸盐缓冲液；发光标记物包括9,10-二氢吖啶标记的SAA捕获抗体和0.05M Tris缓冲液；辅助剂包括发光辅助剂和枸橼酸盐缓冲液。本发明采用试剂空间邻近发光分析检测技术,其作为一种真正意义上的均相化学发光技术,无需清洗、无需载体,没有包被过程,检测灵敏度高,特异性强,使得检测结果更真实可信；同时优化了反应时间和反应步骤,使操作更简单。(The invention relates to a kit for detecting serum amyloid A by a spatial proximity chemiluminescence method and a detection method thereof. The kit comprises: enzyme label, luminescent label, adjuvant, trigger and calibrator; wherein the calibrator comprises calibrators of different concentrations of SAA antigen and 0.1M phosphate diluent; the enzyme label comprises peroxidase-labeled SAA detection antibody and 0.05M phosphate buffer solution; luminescent markers including 9, 10-two hydrogen acridine labeled SAA capture antibody and 0.05M Tris buffer; the adjuvants include luminescence adjuvant and citrate buffer. The invention adopts reagent space proximity luminescence analysis detection technology, which is a true homogeneous phase chemiluminescence technology, and has the advantages of no need of cleaning, no need of carrier, no coating process, high detection sensitivity and strong specificity, thus leading the detection result to be more real and credible; meanwhile, the reaction time and the reaction steps are optimized, so that the operation is simpler.)

1. The application of the serum amyloid A detection kit in the detection of serum amyloid A by a spatial proximity chemiluminescence method is characterized in that the serum amyloid A kit comprises:

enzyme label, luminescent label, adjuvant, trigger and calibrator.

2. The use according to claim 1,

the raw material components of the enzyme marker comprise peroxidase-labeled SAA detection antibodies, and the raw material components of the luminescent marker comprise 9, 10-dihydroacridine-labeled SAA capture antibodies;

the raw material components of the enzyme marker also comprise 0.05M phosphate buffer solution;

the preparation method of the enzyme label comprises the following steps: weighing 5mg of HRP, dissolving in 1mL of distilled water, adding 0.2mL of newly prepared 0.1M sodium periodate solution, stirring at room temperature in a dark place for 20min, filling the solution into a dialysis bag, dialyzing against 1mM sodium acetate buffer solution with pH4.4, and standing at 4 ℃ overnight; adding 20 μ L of 0.2M pH 9.5 carbonate buffer solution, immediately adding 1mg of anti-SAA monoclonal antibody, stirring gently at room temperature in the dark for 2h, adding newly prepared 4mg/mL sodium borohydride solution, mixing, standing at 4 deg.C for 2h, filling the above solution into a dialysis bag, dialyzing against 0.15M pH7.4PBS, and standing at 4 deg.C overnight; taking out the dialyzate, adding appropriate amount of glycerol, and storing in a refrigerator at-20 deg.C;

the raw material component of the luminescent marker also comprises 0.05MTris buffer solution;

the preparation method of the luminescent marker comprises the following steps:

the luminescent substrate Acridan was dissolved in 500. mu.L of DMF;

sucking 41.3 mu L of dissolved Acridan, adding 708.7 mu L of 0.05M sodium borate buffer solution, adding 250 mu L of anti-SAA monoclonal antibody, turning and uniformly mixing for 4-5 times, and standing for 30min at room temperature;

and (3) placing the marked reaction tube on a shaking table, mixing at 2-8 ℃ overnight, taking out, adding a proper amount of glycerol, and placing in a refrigerator at-20 ℃ for storage.

3. The use according to claim 1,

the calibrator further comprises calibrators of different concentrations of SAA antigen and a calibrator diluent;

the preparation method of the calibrator diluent comprises the following steps:

weighing 14.1g of monopotassium phosphate and 3.0g of NaHPO & 2HO, adding ultrapure water for dissolving, adding Proclin-3000.5-1 mL, uniformly mixing, adding ultrapure water for constant volume to 1000mL to obtain a calibrator diluent, and storing at 2-8 ℃ for later use;

the preparation method of the calibrator comprises the following steps:

the concentration of the calibrator is 0, 15ng/mL, 45ng/mL, 135ng/mL, 400ng/mL or 800ng/mL, the purified SAA is diluted to the corresponding concentration by the calibrator diluent, and the purified SAA is stored at the temperature of 2-8 ℃ for later use.

4. The use according to any one of claims 1 to 3, wherein the detection kit is used for detecting serum amyloid A by a spatial proximity chemiluminescence method, and comprises the following steps:

s1: respectively adding 25 mu L of standard substance, 25 mu L of peroxidase-labeled SAA detection antibody and 25 mu L of luminescent marker into a chemiluminescence reaction tube;

s2: reacting for 15min in a constant temperature box at 37 ℃;

s3: respectively adding 5 mu L of auxiliary agent into a chemiluminescence reaction tube, shaking and uniformly mixing, and standing for 1-2 min; then respectively adding 75 mu L of trigger, shaking and uniformly mixing, immediately detecting, and reading a signal value;

s4: and performing logistic four-parameter fitting on the concentration and the luminous value of the calibrator, and calculating the concentration of the sample according to the luminous value of the sample.

Technical Field

The invention relates to the technical field of biology, in particular to a kit for detecting serum amyloid A by a spatial proximity chemiluminescence method and a detection method thereof.

Background

Infectious diseases are common diseases and frequently encountered diseases in clinic, mainly are inflammatory reactions caused by invasion of pathogens such as bacteria, viruses and/or fungi into bodies, and are mostly accompanied by fever symptoms. While many patients are treated and saved their lives, the improper use and abuse of antibacterial drugs has led to numerous adverse consequences, such as extensive and severe bacterial resistance.

The serum amyloid is polypeptide consisting of 104 amino acids, the relative molecular mass of the serum amyloid in a natural state is about 12000-14000, the gene of the serum is located in chromosome 11, a sensitive inflammation marker discovered in recent years is acute phase reaction protein, the concentration of the serum amyloid is obviously increased in acute and chronic inflammatory reactions and can reach more than 1000 times of the normal concentration; meanwhile, the early stage of bacterial and viral infection can be obviously increased, and the early stage of bacterial and viral infection can be quickly increased under stress states of wounds, burns and the like.

Serum Amyloid A (SAA) is a cellular inflammatory factor closely related to the occurrence of cardiovascular disease, and the increase of the level is positively correlated with the occurrence of cardiovascular disease, and can predict the end event of cardiovascular disease in early stage. Research has shown that: serum SAA elevation is detectable in bacterial, fungal, viral infections, atherosclerosis, cardiovascular disease, acute transplant rejection, tumors, and the like. Like C-reactive protein (CRP), SAA helps to diagnose inflammation and evaluate its therapeutic efficacy, but in some diseases, such as viral infection, cardiovascular disease, transplant rejection, etc., SAA sensitivity may be higher than C-reactive protein, providing better reference value for clinical diagnosis.

The detection of CRP in combination with SAA can improve the sensitivity of diagnosis of infection. In the virus infectious diseases, the SAA is obviously increased, and the CRP is not increased; thus, SAA can be used as a sensitive indicator for diagnosing viral infections. The SAA is combined with the CRP detection, so that the bacterial and viral infection can be distinguished and diagnosed, a new basis can be provided, the reliability is higher, the curative effect can be dynamically observed, and the clinical medication can be guided. The SAA is combined with CRP detection, and is beneficial to early diagnosis of infantile infectious diseases (neonatal septicemia and sepsis).

At present, ELISA and nephelometry are mainly adopted clinically, and the two methods have narrow linear range, low sensitivity and poor repeatability. In addition, ELISA requires a solid phase carrier (such as a microplate), and the reaction complex is washed after the reaction is completed to remove unbound free components. The operation is complex, and the influence factors are many, so that the detection result is unstable, and the repeatability is poor.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a kit for detecting serum amyloid A by a spatial proximity chemiluminescence method and a detection method thereof. As a true homogeneous phase chemiluminescence technology, the method does not need a carrier, does not have coating and washing processes, and has the advantages of few kit components, high sensitivity, good repeatability and simple operation.

In order to achieve the purpose, the technical scheme provided by the invention is as follows:

in a first aspect, the present invention provides a serum amyloid a detection kit, comprising: enzyme label, luminescent label, adjuvant, trigger and calibrator; wherein, the raw material component of the enzyme marker comprises SAA detection antibody marked by peroxidase, and the raw material component of the luminescent marker comprises SAA capture antibody marked by 9, 10-dihydroacridine.

Preferably, the raw material components of the enzyme marker also comprise 0.05M phosphate buffer solution; more preferably, the preparation method comprises: weighing 5mg of HRP, dissolving in 1mL of distilled water, adding 0.2mL of newly prepared 0.1M sodium periodate solution, stirring at room temperature in a dark place for 20min, filling the solution into a dialysis bag, dialyzing with 1mM pH4.4 sodium acetate buffer solution, and standing at 4 ℃ overnight; adding 20 μ L of 0.2MpH 9.5.5 carbonate buffer solution, immediately adding 1mg of anti-SAA monoclonal antibody, stirring gently at room temperature and dark for 2h, adding newly prepared 4mg/mL sodium borohydride solution, mixing, standing at 4 deg.C for 2h, filling the above solution into dialysis bag, dialyzing against 0.15MpH7.4PBS, and standing at 4 deg.C overnight; taking out the dialyzate, adding appropriate amount of glycerol, and storing in a refrigerator at-20 deg.C.

Preferably, the raw material component of the luminescent marker further comprises 0.05M Tris buffer; more preferably, the preparation method comprises: the luminescent substrate Acridan was dissolved in 500. mu.L of DMF; sucking dissolved Acridan41.3 mu L, adding 708.7 mu L of 0.05M sodium borate buffer solution, adding 250 mu L of anti-SAA monoclonal antibody, turning and uniformly mixing for 4-5 times, and standing for 30min at room temperature; and (3) placing the marked reaction tube on a shaking table, mixing at 2-8 ℃ overnight, taking out, adding a proper amount of glycerol, and storing in a refrigerator at-20 ℃.

Preferably, the components of the adjuvant include a luminescence adjuvant and a citrate buffer at pH 6.0; more preferably, the preparation method comprises: weighing 1.82g of citric acid and 10.45g of sodium citrate, adding pure water to dissolve and fix the volume to 1000mL, adding a proper amount of luminescence auxiliary agent into citrate buffer solution, mixing uniformly, subpackaging, and placing 10mL of each bottle in a refrigerator at 4 ℃ for later use.

Preferably, the trigger is 0.05MpH 8.0.0 Tris-HCl buffer solution; more preferably, the preparation method comprises: weighing 6.06g of Tris and 9g of sodium chloride, adding a proper amount of pure water for dissolution, adding 2.1mL of concentrated HCl, and uniformly mixing. Adding tween-202 mL into the solution, mixing, diluting to 1000mL, mixing, packaging, and storing in a refrigerator at 4 deg.C for use, wherein 200mL per bottle.

Preferably, the calibrators comprise calibrators of different concentrations of SAA antigen and 0.1M phosphate buffer; more preferably, the preparation method comprises: preparing a calibrator diluent: weighing 14.1g of monopotassium phosphate and 3.0g of sodium dihydrogen phosphate (2H 2O), adding a proper amount of ultrapure water for dissolving, namely Proclin-3000.5-1 mL, uniformly mixing, adding ultrapure water for constant volume to 1000mL, namely, preparing a calibrator diluent, and storing at 2-8 ℃ for later use; preparing a calibrator: the concentration of the calibrator is 0, 15ng/mL, 45ng/mL, 135ng/mL, 400ng/mL and 800ng/mL, the purified serum amyloid A is diluted to the corresponding concentration by the calibrator diluent, and the calibrator is stored at 2-8 ℃ for later use.

The kit provided by the invention adopts a double-antibody sandwich method to detect the content of serum amyloid protein A (SAA) in human serum. Reacting a sample, an anti-SAA monoclonal antibody marked by horseradish peroxidase (HRP) and an anti-SAA monoclonal antibody marked by 9, 10-dihydroacridine (Acridan) together to form an antigen-antibody sandwich complex, enabling the horseradish peroxidase and the 9, 10-dihydroacridine (Acridan) to be close to each other in space, and adding a luminescent auxiliary agent and a trigger to generate flash chemiluminescence; unbound free HRP-labeled antibody and Acridan-labeled antibody did not emit light. The higher the amount of SAA in the sample, the greater the luminescence value (RLU) measured. Therefore, within a certain concentration range, the luminous value is in positive correlation with the concentration of the sample, and the content of the SAA in the sample can be calculated according to the luminous value of the sample by drawing a working curve through the calibrator with known concentration and the luminous value thereof.

In a second aspect, the invention provides the use of the detection kit in detecting serum amyloid A by a spatial proximity chemiluminescence method.

In a third aspect, the present invention provides a method for detecting serum amyloid a by using the above detection kit in a spatial proximity chemiluminescence method, comprising the steps of:

s1: respectively adding 25 mu L of standard substance, 25 mu L of peroxidase-labeled SAA detection antibody and 25 mu L of luminescent marker into the reaction tube;

s2: reacting for 15min in a constant temperature box at 37 ℃; s3: respectively adding 5 mu L of auxiliary agent into the reaction tube, shaking and uniformly mixing, and standing for 1-2 min; then respectively adding 75 mu L of trigger, shaking and uniformly mixing, immediately detecting, and reading a signal value;

s4: and performing logistic four-parameter fitting on the concentration and the luminous value of the calibrator, and calculating the concentration of the sample according to the luminous value of the sample.

The technical scheme provided by the invention has the following beneficial effects:

(1) compared with the traditional chemiluminescence technology which needs a microporous plate or magnetic particles as a carrier to coat an antibody or an antigen, the kit and the detection method provided by the invention do not need a carrier and do not have coating and washing processes, and are a true homogeneous chemiluminescence technology.

(2) In the traditional enzymatic chemiluminescence technology, an analyte to be detected is combined with a capture antibody and an enzyme-labeled detection antibody in sequence and then is washed for 2-3 times to remove unbound or loosely bound non-specific substances; in the invention, after the analyte to be detected is combined with the two specific antibodies, the luminescence interference is eliminated through the auxiliary agent effect, and the whole process of adding the trigger to generate the luminescence signal does not need washing.

(3) The traditional enzymatic chemiluminescence technology needs an enzyme catalysis substrate, and the luminescence value is detected in about 5-10 minutes; the invention is a flash-type chemiluminescence, which generates a luminescent signal immediately after a trigger is added.

(4) The kit provided by the invention has the advantages of few components, simple production process, low production cost and easy amplification production; and the detection process is convenient and fast, and the full automation is easy to realize.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Detailed Description

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.

The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. In the quantitative tests in the following examples, three replicates were set, and the data are the mean or the mean ± standard deviation of the three replicates.

The invention provides a detection kit for serum amyloid A, which comprises: enzyme label, luminescent label, adjuvant, trigger and calibrator; wherein, the raw material components of the enzyme label comprise peroxidase-labeled SAA detection antibody and 0.05M phosphate buffer solution; the raw material components of the luminescent marker comprise a 9, 10-dihydroacridine marked SAA capture antibody and a 0.05M Tris buffer solution; the raw material components of the auxiliary agent comprise a luminescence auxiliary agent and a citrate buffer solution with the pH value of 6.0; the trigger is 0.05M Tris-HCl buffer solution with the pH value of 8.0; the calibrator included calibrators of different concentrations of SAA antigen and 0.1M dilution of the calibrator.

Preparation of calibrator

(1) Preparing a calibrator diluent: 14.1g of potassium dihydrogen phosphate and sodium dihydrogen phosphate (2H) were weighed₂O)3.0g, adding ultrapure water for dissolving, Proclin-3000.5-1 mL, mixing uniformly, adding ultrapure water for constant volume to 1000mL to obtain a calibrator diluent, and storing at 2-8 ℃ for later use.

(2) Preparing a calibrator: the concentration of the calibrator is 0, 15ng/mL, 45ng/mL, 135ng/mL, 400ng/mL and 800ng/mL, the purified serum amyloid A is diluted to the corresponding concentration by the calibrator diluent, and the calibrator is stored at 2-8 ℃ for later use.

Secondly, preparation of enzyme label

(1) 5mg of HRP was weighed and dissolved in 1mL of distilled water, 0.2mL of newly prepared 0.1M sodium periodate solution was added to the supernatant, and the mixture was stirred at room temperature in the dark for 20min, and the solution was packed in a dialysis bag and dialyzed against 1mM pH4.4 sodium acetate buffer at 4 ℃ overnight.

(2) Adding 20 μ L of 0.2MpH 9.5.5 carbonate buffer solution, immediately adding 1mg of anti-SAA monoclonal antibody, stirring gently at room temperature and dark for 2h, adding new 4mg/mL sodium borohydride solution, mixing, standing at 4 deg.C for 2h, filling the above solution into dialysis bag, dialyzing against 0.15MpH 7.4.4 PBS, and standing at 4 deg.C overnight.

(3) Taking out the dialyzate, adding appropriate amount of glycerol, and storing in a refrigerator at-20 deg.C.

Preparation of luminescent markers

(1) The luminogenic substrate Acridan was dissolved in 500. mu.L of DMF.

(2) Sucking dissolved Acridan41.3 mu L, adding 0.05M sodium borate buffer 708.7 mu L, adding 250 mu L anti-SAA monoclonal antibody, turning and uniformly mixing for 4-5 times, and standing for 30min at room temperature.

(3) And (3) placing the marked reaction tube on a shaking table, mixing at 2-8 ℃ overnight, taking out, adding a proper amount of glycerol, and storing in a refrigerator at-20 ℃.

Preparation of adjuvant

Weighing 1.82g of citric acid and 10.45g of sodium citrate, adding pure water to dissolve and fix the volume to 1000mL, adding a luminescence auxiliary agent into a citrate buffer solution, mixing uniformly, subpackaging, and storing in a refrigerator at 4 ℃ for later use; among them, 10mL per bottle is more preferable.

Preparation of trigger

Weighing 6.06g of Tris and 9g of sodium chloride, adding a proper amount of pure water for dissolving, adding 2.1mL of concentrated HCl, and uniformly mixing; adding tween-202 mL, mixing, diluting to 1000mL, packaging, and storing in a refrigerator at 4 deg.C; among them, more preferably 200mL per bottle.

The invention also provides a method for detecting serum amyloid A by adopting the serum amyloid A detection kit in a space proximity chemiluminescence method, which comprises the following steps:

s1: add 25. mu.L of standard, 25. mu.L of peroxidase-labeled SAA detection antibody, and 25. mu.L of luminescent label, respectively, to the chemiluminescent reaction tube.

S2: the reaction was carried out at 37 ℃ for 15 min.

S3: respectively adding 5 mu L of auxiliary agent into a chemiluminescence reaction tube, shaking and uniformly mixing, and standing for 1-2 min; then, 75 mu L of trigger is added respectively, the mixture is shaken and mixed evenly, and the signal value is read immediately.

S4: and performing logistic four-parameter fitting on the concentration and the luminous value of the calibrator, and calculating the concentration of the sample according to the luminous value of the sample.

The technical solution provided by the present invention is further illustrated below with reference to specific examples.

Example one

This embodiment provides a detection kit for serum amyloid a, including: enzyme label, luminescent label, adjuvant, trigger and calibrator; wherein the calibrator comprises not less than 6 calibrators of SAA antigens with different concentrations and 0.1M phosphate buffer solution; the enzyme label comprises peroxidase-labeled SAA detection antibody and 0.05M phosphate buffer solution; luminescent markers including 9, 10-two hydrogen acridine labeled SAA capture antibody and 0.05M Tris buffer; the adjuvant comprises a luminescence adjuvant and a citrate buffer solution with pH of 6.0; the trigger is 0.05MpH 8.0.0 Tris-HCl buffer solution.

Preparation of calibrator

(1) Preparing a calibrator diluent: 14.1g of potassium dihydrogen phosphate and sodium dihydrogen phosphate (2H) were weighed₂O)3.0g, adding ultrapure water for dissolving, Proclin-3000.8mL, mixing uniformly, adding ultrapure water for constant volume to 1000mL to obtain a calibrator diluent, and storing at 4 ℃ for later use.

(2) Preparing a calibrator: the concentration of calibrator is 0, 15ng/mL, 45ng/mL, 135ng/mL, 400ng/mL, 800ng/mL, and purified serum amyloid A is diluted to corresponding concentration with calibrator diluent, and stored at 4 ℃ for use.

Secondly, preparation of enzyme label

(1) Weighing 5mg of HRP, dissolving in 1mL of distilled water, adding 0.2mL of newly prepared 0.1M sodium periodate solution into the upper solution, stirring at room temperature in a dark place for 20min, filling the solution into a dialysis bag, dialyzing against 1mM pH4.4 sodium acetate buffer solution, and standing at 4 ℃ overnight;

(2) adding 20 μ L of 0.2MpH 9.5.5 carbonate buffer solution, immediately adding 1mg of anti-SAA monoclonal antibody, stirring gently at room temperature and dark for 2h, adding newly prepared 4mg/mL sodium borohydride solution, mixing, standing at 4 deg.C for 2h, placing the above solution in a dialysis bag, dialyzing against 0.15MpH 7.4.4 PBS, and standing at 4 deg.C overnight;

(3) taking out the dialyzate, adding appropriate amount of glycerol, and storing in a refrigerator at-20 deg.C.

Preparation of luminescent markers

(1) The luminescent substrate Acridan was dissolved in 500. mu.L of DMF;

(2) sucking dissolved Acridan41.3 mu L, adding 0.05M sodium borate buffer solution 708.7 mu L, adding 250 mu L anti-SAA monoclonal antibody, turning and mixing uniformly for 5 times, and standing at room temperature for 30 min;

(3) the labeled reaction tube was placed on a shaker, mixed overnight at 4 ℃ and taken out, added with an appropriate amount of glycerol and stored in a refrigerator at-20 ℃.

Preparation of adjuvant

Weighing 1.82g of citric acid and 10.45g of sodium citrate, adding pure water to dissolve and fix the volume to 1000mL, adding a luminescence auxiliary agent into a citrate buffer solution, uniformly mixing, subpackaging, and storing 10mL of each bottle in a refrigerator at 4 ℃ for later use.

Preparation of trigger

Weighing 6.06g of Tris and 9g of sodium chloride, adding a proper amount of pure water for dissolving, adding 2.1mL of concentrated HCl, and uniformly mixing; adding tween-202 mL, mixing, diluting to 1000mL, packaging, and storing 200mL per bottle in a refrigerator at 4 deg.C for use.

Example two

The embodiment provides a method for detecting serum amyloid A by adopting a serum amyloid A detection kit in a space proximity chemiluminescence method, which comprises the following steps:

s1: add 25. mu.L of standard, 25. mu.L of peroxidase-labeled SAA detection antibody, and 25. mu.L of luminescent label, respectively, to the chemiluminescent reaction tube.

S2: the reaction was carried out at 37 ℃ for 15 min.

S3: respectively adding 5 mu L of auxiliary agent into a chemiluminescence reaction tube, shaking and uniformly mixing, and standing for 1-2 min; then, 75 mu L of trigger is added respectively, the mixture is shaken and mixed evenly, and the signal value is read immediately.

S4: and performing logistic four-parameter fitting on the concentration and the luminous value of the calibrator, and calculating the concentration of the sample according to the luminous value of the sample.

The invention adopts a space proximity chemiluminescence method to detect serum amyloid A, and the sensitivity can reach 1 mg/L. The SAA detection result has higher clinical significance for diagnosing virus infection, and the result shows that: the SAA results were significantly elevated in 99% Echo-30 viral meningitis, 97% measles, 95% mumps, while 56% of CRP results were normal in the acute stage of children.

Of course, other conditions and parameters in the preparation process are possible in addition to those recited in example one and example two.

The applicant finds out after creative work that: as a true homogeneous phase chemiluminescence technology, the detection method provided by the invention does not need a carrier, does not have coating and washing processes, and has the advantages of few kit components, high sensitivity, good repeatability and simple operation.

It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains. Unless specifically stated otherwise, the relative steps, numerical expressions, and values of the components and steps set forth in these embodiments do not limit the scope of the present invention. In all examples shown and described herein, unless otherwise specified, any particular value should be construed as merely illustrative, and not restrictive, and thus other examples of example embodiments may have different values.

In the description of the present invention, it is to be understood that the terms "first", "second" and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implying any number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention, and all of the technical solutions are covered in the protective scope of the present invention.