CN103018464B

CN103018464B - Reagent for determining procalcitonin and preparation method of reagent

Info

Publication number: CN103018464B
Application number: CN201210536453.7A
Authority: CN
Inventors: 陈开华; 其他发明人请求不公开姓名
Original assignee: Yuensun Biological Technology (shanghai) Co Ltd
Current assignee: Yuensun Biological Technology (shanghai) Co Ltd
Priority date: 2012-12-12
Filing date: 2012-12-12
Publication date: 2015-01-07
Anticipated expiration: 2032-12-12
Also published as: CN103018464A

Abstract

The invention relates to a reagent for determining procalcitonin and a preparation method of the reagent, in order to provide the reagent which has the characteristic of high accuracy degree, and the provided preparation method has the characteristic of convenience in preparation. The technical scheme is that the reagent for determining procalcitonin comprises the following components: a. a procalcitonin reagent 1, b. a procalcitonin reagent 2, and c. a liquid-type procalcitonin reference calibration item. The preparation method of the reagent for determining procalcitonin comprises the following steps of: (1) uniformly mixing the procalcitonin reagent 1; (2) removing the procalcitonin reagent 2, the removing process specifically comprises the following steps of: a. taking a suspension, b. carrying out reaction on a mixture, c. acquiring a carboxylic latex microsphere suspension, d. regulating the concentration, e. taking the suspension in step (3), and adding into the step (4), f. reacting, g. adding cholamine, and h. carrying out centrifugal treatment; and (3) treating the liquid-type procalcitonin reference calibration product: mixing according to the amount of a formula, and ranking according to the content of the procalcitonin or adding a procalcitonin pure product in the mixed liquid.

Description

Reagent for determining procalcitonin and preparation method of reagent

Technical Field

The invention relates to a reagent for measuring procalcitonin and a preparation method thereof, in particular to a reagent for measuring procalcitonin in serum or plasma by adopting a latex enhanced immunoturbidimetry and a preparation method thereof, which can be widely applied to the technical field of medicine and biochemistry.

Background

Procalcitonin (PCT) is a glycoprotein consisting of 116 amino acids and having a molecular weight of 13 KD. The half-life is 25-30 hours. The PCT content of the plasma of a healthy person is extremely low and is lower than 0.5ng/ml in the blood of a normal person. Its levels in plasma increase when severe bacterial, fungal, parasitic infections and sepsis and multi-organ failure; but the PCT level can not be increased when the immunity, the allergy and the PCT are applied to the diseases such as local infection, virus infection, chronic nonspecific inflammation, cancer fever, graft-host rejection reaction and the like; this determines the high specificity of PCT and can therefore also be used for differential diagnosis in a variety of clinical situations. PCT concentration and inflammation severity are positively correlated and reduced to normal levels with inflammation control and disease remission, so that PCT can be used as a reliable index for disease and prognosis judgment and curative effect observation. PCT can be detected 2 hours after infection, has important significance for clinical early diagnosis, and reaches a peak 12-24 hours after infection. PCT has wide and important application value in clinic; and provide criteria for mortality and length of stay in ICU patients.

The current laboratory methods commonly used for detecting PCT include Radioimmunoassay (RIA), chemiluminescence immunoassay (CLIA), colloidal gold colorimetry (GICA) and enzyme-linked immunosorbent assay (ELISA), wherein the radioimmunoassay is a method for specifically recognizing and connecting artificially synthesized polyclonal antibodies to synthesize the amino acid procalcitonin. The method can detect serum PCT of normal people with reliable sensitivity of 4pm/mL, and detect the mixture of free PCT, bound PCT and calcitonin gene-related peptide precursor, but cannot distinguish the three substances. The method has long detection time (19-22 h), radioactive element pollution, poor marker stability and difficult waste treatment, so that the method is limited. Chemiluminescence immunoassay (CLIA) is a method that chemiluminescence is catalyzed by a catalyst and oxidized by an oxidant to form an excited intermediate; when the intermediate in the excited state returns to the stable ground state, photons are emitted, and the number of photons is measured by using a luminescence signal measuring instrument, so that the concentration of PCT is indirectly measured. At present, tubular and plate kits are available on the market, but the method has the defects of low luminous efficiency and unstable marker of the acridinium ester, luminol and isoluminol direct labeled antibody; moreover, the direct marking method belongs to an instant luminous type, so that the stability and the repeatability of a test result are difficult to ensure, and a special detection instrument is required. Is inconvenient for routine laboratory development. The electrochemical luminescence immunoassay kit for detecting PCT by using the biotin-avidin system needs to be matched with an expensive full-automatic electrochemical luminescence detector, cannot be developed in a conventional laboratory, brings unnecessary cost to a patient and increases the burden of the patient. The enzyme linked immunosorbent assay is characterized in that an artificially synthesized PCT monoclonal antibody is used for detection, although the assay method is specific, the minimum detection limit is 10 mu g/L, and the concentration of PCT in the serum of a normal person cannot be detected. Chinese patent (application No. 201110210250.4) also discloses a kit for detecting procalcitonin, which utilizes a magnetic particle coupled antibody as a fixed separation phase, a biotin-streptavidin multistage amplification system and horseradish peroxidase as a catalytic enzyme catalytic luminescent substrate, so that although the detection sensitivity is improved, the whole reaction takes longer time (at least 40 minutes), and a sample needs magnetic separation pretreatment. The method needs to wash the reaction tube, if the reaction tube is washed manually, human errors can be caused, if the reaction tube is washed by an instrument, the instrument needs to be additionally prepared, and the cost of a laboratory is increased.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects of the background technology and provide a procalcitonin detection reagent and a preparation method thereof; the provided reagent has the characteristics of high accuracy, good repeatability, simple operation, short determination time and suitability for various types of full-automatic biochemical analyzers; the provided reagent preparation method has the characteristics of convenient manufacture and low cost.

The technical scheme provided by the invention is as follows:

a reagent for determining procalcitonin comprises the following components in parts by weight:

a. procalcitonin reagent 1:

the pH value of the reagent 1 is 7.0-8.0;

b. procalcitonin reagent 2:

the particle size of the carboxyl latex microspheres in the anti-human PCT antibody latex particles is 20nm-500 nm;

c. liquid procalcitonin reference calibrator:

the liquid procalcitonin reference calibrator comprises 5 reference calibrators with a series of concentrations; the procalcitonin content in the 5 reference calibrators is respectively 1.25ng/ml, 2.5ng/ml, 5.0ng/ml, 10.0ng/ml and 20.0ng/ml from low to high; or preparing a procalcitonin reference calibrator with single-point concentration; for example a procalcitonin reference calibrator at a concentration of 100ng/ml or 500 ng/ml.

The buffer solution is one or a mixture of more than two of phosphate buffer solution, Tris buffer solution, 1, 4-piperazine disulfonic acid (PIPES) buffer solution, 4- (2-hydroxyethyl) piperazine-1-1 ethane sulfonic acid (HEPES) buffer solution, Tris buffer solution, glycine buffer solution, boric acid buffer solution, 3-morpholine propanesulfonic acid (MOPS) buffer solution, 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid (MOPSO) buffer solution and 2-morpholine ethanesulfonic acid (MES) buffer solution in any proportion.

The concentration of the buffer solution in the procalcitonin reagent 1 is 20-200mmol/L, and the pH value is 7.0-8.0.

The reaction accelerator is polyethylene glycol or dextran sulfate (DS-50).

The concentration of the reaction accelerator is preferably 0.05 to 30 g/L.

The stabilizer is one or a mixture of more than two of ethylene diamine tetraacetic acid, bovine serum albumin and sodium chloride in any proportion.

The electrolyte is an anionic electrolyte or a cationic electrolyte.

The electrolyte is sodium chloride (NaCl) in the cation, preferably in a concentration of 50-200 mmol/L.

The surfactant is a nonionic surfactant, a cationic surfactant, an anionic surfactant, or a zwitterionic surfactant.

The nonionic surfactant is: theist, Tween series, polyoxyethylene lauryl ether series, polyoxyethylene phenyl ether, polyoxyethylene octyl phenyl ether, polyoxyethylene alkyl phenyl ether and polyoxyethylene nonyl phenyl ether in any proportion.

The concentration of the surfactant is preferably 0.5 to 1 g/L.

The concentration of the anti-human PCT antibody latex particles is preferably 1.0-10 mg/ml.

The anti-human procalcitonin antibody is a goat anti-human procalcitonin antibody, a rabbit anti-human procalcitonin antibody, a chicken anti-human procalcitonin antibody or a mouse anti-human procalcitonin antibody.

A preparation method of a procalcitonin determination reagent comprises the following steps:

1) procalcitonin reagent 1:

uniformly mixing a buffer solution, an electrolyte, a surfactant, a reaction accelerator, a preservative, a stabilizer and purified water according to the formula amount, and adjusting the pH value to 7.0-8.0;

2) procalcitonin reagent 2:

(1) diluting carboxyl latex microspheres into 0.01mg/ml suspension by using buffer solution;

(2) adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide sulfonic acid sodium salt (NHS) in a ratio of 20mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC carboxyl activator) and 40mg of N-hydroxysuccinimide sulfonic acid sodium salt (NHS) to 1ml of carboxyl latex microsphere suspension, immediately mixing uniformly, reacting the mixture at room temperature for 15-30 minutes, and continuously stirring;

(3) washing carboxyl latex microspheres by using buffer solution or purified water, removing unreacted 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide sulfonic acid sodium salt, and suspending the latex microspheres in the purified water to obtain carboxyl latex microsphere suspension with the concentration of 0.01 mg/ml;

(4) dissolving the anti-human procalcitonin antibody in a buffer solution to ensure that the protein concentration of the anti-human procalcitonin antibody is 0.25 mg/ml;

(5) taking 1ml of the carboxyl latex microsphere suspension in the step (3), and immediately adding 1ml of the antihuman procalcitonin antibody in the step (4); the concentrations of the carboxyl latex microspheres, the antihuman procalcitonin antibody and the buffer solution in the mixed solution are respectively 0.005mg/ml, 0.125mg/ml and 25 mmol/L;

(6) reacting the mixture at 37 ℃ for 3 hours, and continuously stirring;

(7) adding ethanolamine into 1ml of the reaction mixture in the ratio of 2.5 microliter ethanolamine, reacting for 10-30 min and stirring;

(8) centrifuging to remove unbound protein and ethanolamine, diluting with buffer solution to make the concentration of antihuman PCT antibody latex particles be 0.002-0.01ml/ml, adding stabilizer and antiseptic, dissolving, and mixing to obtain procalcitonin reagent 2;

3) liquid procalcitonin reference calibrator:

firstly, mixing a preservative, a stabilizer and a buffer solution according to the formula amount, dividing the mixture into 5 parts of mixed liquor, and then respectively adding 1000ng/ml of procalcitonin pure product with corresponding amount into the mixed liquor according to the required procalcitonin reference calibrator concentration to obtain 5 parts of reference calibrators with serial concentrations; the procalcitonin content in the 5 reference calibrators is respectively 1.25ng/ml, 2.5ng/ml, 5.0ng/ml, 10.0ng/ml and 20.0ng/ml from low to high; or,

mixing the preservative, the stabilizer and the buffer solution according to the formula amount to obtain a mixed solution, and then adding a certain amount of 1000ng/ml procalcitonin pure product into the mixed solution to obtain the single-point high-concentration procalcitonin reference calibrator. For example, the reference calibrator may contain procalcitonin in an amount of 100.0ng/ml or 500.0 ng/ml; when in use, the standard substance is diluted into a plurality of reference standard substances with different concentrations by using normal saline; there is no particular limitation as long as the prepared procalcitonin reference calibrator can be compared with a sample and the content of procalcitonin in the sample can be determined.

The centrifugal treatment adopts a centrifugal machine, and the rotating speed of the centrifugal machine is 15000 r/min.

The reaction principle of the PCT determination reagent provided by the invention is as follows: the PCT in the sample and the anti-human PCT antibody latex particles in the reagent generate antigen-antibody reaction to increase the turbidity of the reaction solution, the turbidity of the reaction solution and the amount of the antigen in the sample are in a linear relation in a certain range, a biochemical analyzer or other optical detection instruments can be used for measuring the absorbance value of the reaction solution at the wavelength of 600nm at 520 ℃, and the absorbance value of the reaction solution is in direct proportion to the concentration of the PCT to be measured.

The invention has the beneficial effects that: the provided procalcitonin detection reagent adopting the latex enhanced immunoturbidimetry amplifies the surface area of reaction of the CG antigen and the antibody by combining the CG antibody with the carboxyl latex microspheres, and has high accuracy (correlation R with a chemiluminescence immunoassay method)²0.9991-0.9998), good repeatability and quick detection (the result is obtained only needs 10 minutes at most from the beginning of the detection), can carry out large-batch sample detection on a conventional biochemical analyzer, and greatly improves the detection working efficiency. The preparation method of the reagent is simple and convenient to operate, the raw materials are easy to obtain (all outsourcing raw materials are selected), the cost is low, and the reagent is suitable for various medical research units and conventional laboratories.

Drawings

FIG. 1 shows the correlation between the measured value of the PCT reagent obtained by the method of the present invention and the value of PCT obtained by the chemiluminescence immunoassay in example 1. Through regression analysis: y =1.0062x + 0.6087; r²=0.9991, showing that example 1 has good correlation with chemiluminescence immunoassay.

FIG. 2 shows the correlation between the measured value of the PCT reagent obtained by the method of the present invention and the value of PCT obtained by the chemiluminescence immunoassay in example 2. Through regression analysis: y =1.0096x + 0.7981; r²=0.9996, demonstrating this example 2 and chemiluminescence immunoassayThe assay has good correlation.

FIG. 3 shows the correlation between the measured value of the PCT reagent obtained by the method of the present invention and the value of PCT obtained by the chemiluminescence immunoassay in example 3. Through regression analysis: y =1.0092x + 0.7507; r²=0.9998, showing that this example 3 has a good correlation with the chemiluminescence immunoassay.

FIG. 4 shows the correlation between the measured value of the PCT reagent obtained by the method of the present invention and the value of PCT obtained by the chemiluminescence immunoassay in example 4. Through regression analysis: y =1.0056x + 2.8301; r²=0.9998 shows that this example 4 has a good correlation with the chemiluminescence immunoassay.

Detailed Description

The detection reagent and the calibrator of the invention require the following main raw materials:

1. as anti-human procalcitonin antibodies, there are many commercially available anti-human procalcitonin antibodies available from, for example, the Finland DaKo company, the Japanese UNF company; the antibody reacts only with human procalcitonin, has no immunological cross reaction with other antigens, can be a polyclonal antibody or a monoclonal antibody, and is not particularly limited as long as the antibody can be coupled with latex microspheres. The anti-human procalcitonin antibody can be goat antibody (i.e. goat anti-human procalcitonin antibody; the same applies below), rabbit antibody, chicken antibody or mouse antibody. The antibody exhibits only a single sedimentation line with human PCT, and the anti-human procalcitonin antibody titer is greater than or equal to 0.5mg/ml, as long as it is determined by known immunoelectrophoresis.

2. Latex microspheres, there are many commercially available nanospheres available, such as Merck, Japan UNF, JSR, and Thermo; the basic material is polystyrene or copolymerized styrene, the surface of the polystyrene or copolymerized styrene is modified by carboxyl groups (COOH), and the polystyrene or copolymerized styrene can be used without any modification, wherein different microspheres are selected according to different combination methods of selected antibodies and microspheres. If a chemical crosslinking method (physical adsorption method is also available), microspheres modified with carboxyl, amino, aldehyde groups, etc. on their surface are used. The method is preferably carried out on latex microspheres modified by carboxyl groups, and the particle size of the carboxyl latex microspheres is preferably 20nm-500nm, more preferably 80nm-300 nm.

3. Procalcitonin: the standard substance which is purchased from DaKo company in Finland and is used for preparing the reagent can be natural procalcitonin or gene recombinant procalcitonin.

The reagents of the present invention are described in detail below with reference to examples, but these examples should not be construed as limiting the definition of the present invention.

Example one

One) Procalcitonin reagent 1

Dissolving the raw materials by using 800ml of purified water, supplementing the purified water to a constant volume of 1000ml, and then adjusting the pH value to 7.2 by using hydrochloric acid;

II) Procalcitonin reagent 2

1. Diluting 80nm carboxyl latex microspheres with 50mmol/L MES buffer solution (pH6.0) to obtain 0.01mg/ml suspension;

2. adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide sulphonic acid sodium salt in a proportion of 20mg 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) and 40mg N-hydroxysuccinimide sulphonic acid sodium salt (NHS) to 1ml of carboxyl latex microsphere suspension, immediately mixing well, reacting the mixture at room temperature for 15-30 minutes, and stirring continuously;

3. washing the carboxyl latex microspheres with 50mmol/L, PH 6.0.0 MES buffer solution or purified water to remove unreacted NHS and EDAC, and suspending the latex microspheres in purified water to obtain a carboxyl latex microsphere suspension, wherein the content of the carboxyl latex microspheres is 0.01 mg/ml;

4. dissolving the goat anti-human procalcitonin antibody in 50mmol/L, PH 8.5.5 of borate buffer solution to ensure that the protein concentration of the goat anti-human procalcitonin antibody is 0.25 mg/ml;

5. taking 1ml of the carboxyl latex microsphere suspension in the step (3), and immediately adding 1ml of the goat anti-human procalcitonin antibody in the step (4); the concentrations of the carboxyl latex microspheres, the goat anti-human procalcitonin antibody and the buffer solution in the mixed solution are 0.005mg/ml, 0.125mg/ml and 25mmol/L respectively;

6. reacting the mixture at 37 ℃ for 3 hours, and continuously stirring;

7. adding ethanolamine into 1ml of the reaction mixture according to the proportion of adding 2.5 mul of ethanolamine, reacting for 15 minutes, and continuously stirring;

8. removing unbound protein and ethanolamine by 15000 r/m separation, diluting with 50mmol/L, PH 7.2.2 MOPSO buffer solution to make the concentration of goat anti-human PCT antibody latex particles 0.01ml/ml, adding bovine serum albumin (stabilizer) 3mmol/L and 3mmol/L sodium azide (preservative), dissolving and mixing well to obtain procalcitonin reagent 2;

III) liquid type PCT reference calibration substance

Adding 1000ng/ml of the corresponding PCT pure product into the mixed solution according to the required concentration of the PCT reference calibrator to prepare 5 reference calibrators with serial concentrations; the PCT content of 5 reference calibrators from low to high was 5ng/ml, 10ng/ml, 20ng/ml, 40ng/ml and 80ng/ml respectively.

Sample detection (30 samples were detected in contrast to the chemiluminescence immunoassay, respectively):

according to the experimental procedure, 240. mu.L of procalcitonin reagent 1 was added to 10. mu.L of the sample, incubated at 37 ℃ for 3 minutes, 60. mu.L of procalcitonin reagent 2 was added, and after incubation at 37 ℃ for 10 seconds, the absorbance A was read at a wavelength of 600nm₁And reading the absorbance A when the reaction time is 5 minutes₂Calculating the absorbance change value delta A = A twice₂-A₁. PCT concentration in the sample was calculated by comparison with the same treated calibration solutions. As can be seen from the results in Table 1 and FIG. 1 (FIG. 1 is plotted based on the data in Table 1), the method of the present invention shows very good correlation (correlation R) with the chemiluminescence immunoassay, although the operation is simple and the time required is only 10 minutes or less²To 0.9991).

TABLE 1

Example two

One) Procalcitonin reagent 1

Dissolving the raw materials by using 800ml of purified water, supplementing the purified water to a constant volume of 1000ml, and then adjusting the pH value to 8.0 by using sodium hydroxide;

II) Procalcitonin reagent 2

1. Diluting 150nm carboxyl latex microspheres with 50mmol/L, PH 6.0.0 MES buffer solution to obtain 0.01mg/ml suspension;

2. adding EDAC and NHS according to the proportion of adding 20mg of EDAC and 40mg of NHS into 1ml of carboxyl latex microsphere suspension, immediately mixing uniformly, reacting the mixture for 15-30 minutes at room temperature, and continuously stirring;

4. dissolving rabbit anti-human procalcitonin antibody in 50mmol/L, PH 8.5.5 borate buffer solution to make the protein concentration of rabbit anti-human procalcitonin antibody be 0.25 mg/ml;

5. taking 1ml of the carboxyl latex microsphere suspension in the step (3), and immediately adding 1ml of the rabbit anti-human procalcitonin antibody in the step (4); the concentrations of the carboxyl latex microspheres, the rabbit anti-human procalcitonin antibody and the buffer solution in the mixed solution are 0.005mg/ml, 0.125mg/ml and 25mmol/L respectively;

6. reacting the mixture at 37 ℃ for 3 hours, and continuously stirring;

7. adding ethanolamine into 1ml of the reaction mixture according to the proportion of adding 2.5 mul of ethanolamine, reacting for 30 minutes, and continuously stirring;

8. removing unbound protein and ethanolamine by 15000 r/m.F., diluting with 50mmol/L, PH 7.4.4 phosphate buffer solution to make the concentration of rabbit anti-human PCT antibody latex particles 0.006ml/ml, adding bovine serum albumin (stabilizer) 3mmol/L and sodium azide (preservative) 3mmol/L, dissolving and mixing to obtain procalcitonin reagent 2;

III) liquid type PCT reference calibration substance

And adding 1000ng/ml of the corresponding pure PCT product into the mixed solution according to the required concentration of the PCT reference calibrator to prepare 5 series concentrations of the reference calibrator, wherein the PCT contents of the 5 series concentrations of the reference calibrator are respectively 7.5ng/ml, 15ng/ml, 30ng/ml, 60ng/ml and 120ng/ml from low to high.

according to the experimental procedure, 240. mu.L of procalcitonin reagent 1 was added to 10. mu.L of the sample, incubated at 37 ℃ for 3 minutes, 60. mu.L of procalcitonin reagent 2 was added, and after incubation at 37 ℃ for 10 seconds, the absorbance A was read at a wavelength of 600nm₁And reading the absorbance A when the reaction time is 5 minutes₂Calculating the absorbance change value delta A = A twice₂-A₁. PCT concentration in the sample was calculated by comparison with the same treated calibration solutions. As can be seen from the results of Table 2 and FIG. 2 (FIG. 2 is plotted based on the data of Table 2), the method of the present invention shows very good correlation (correlation R) with the chemiluminescence immunoassay, although the operation is simple and the time required is only within 10 minutes²To 0.9996).

TABLE 2

EXAMPLE III

One) Procalcitonin reagent 1

II) Procalcitonin reagent 2

1. Diluting 300nm carboxyl latex microspheres with 50mmol/L, PH 6.0.0 MES buffer solution to obtain 0.01mg/ml suspension;

4. dissolving the mouse anti-human procalcitonin antibody in 50mmol/L, PH 8.5.5 of borate buffer solution to ensure that the protein concentration of the mouse anti-human procalcitonin antibody is 0.25 mg/ml;

5. taking 1ml of the carboxyl latex microsphere suspension in the step (3), and immediately adding 1ml of the mouse anti-human procalcitonin antibody in the step (4); the concentrations of the carboxyl latex microspheres, the mouse anti-human procalcitonin antibody and the buffer solution in the mixed solution are 0.005mg/ml, 0.125mg/ml and 25mmol/L respectively;

6. reacting the mixture at 37 ℃ for 3 hours, and continuously stirring;

7. adding ethanolamine into 1ml of the reaction mixture according to the proportion of adding 2.5 mul of ethanolamine, reacting for 10 minutes, and continuously stirring;

8. removing unbound protein and ethanolamine by 15000 r/m.c., diluting with 50 mmol/358.0 PIPES buffer solution to make the concentration of mouse anti-human PCT antibody latex particles 0.002ml/ml, adding bovine serum albumin (stabilizer) 3mmol/L and sodium azide (preservative) 3mmol/L, dissolving and mixing to obtain procalcitonin reagent 2.

III) liquid type PCT reference calibration substance

And adding 1000ng/ml of the corresponding pure PCT product into the mixed solution according to the required concentration of the PCT reference calibrator to prepare 5 series concentrations of the reference calibrator, wherein the PCT contents of the 5 series concentrations of the reference calibrator are respectively 10ng/ml, 40ng/ml, 80ng/ml, 160ng/ml and 320ng/ml from low to high.

according to the experimental procedure, 240. mu.L of procalcitonin reagent 1 was added to 10. mu.L of the sample, incubated at 37 ℃ for 3 minutes, 60. mu.L of procalcitonin reagent 2 was added, and after incubation at 37 ℃ for 10 seconds, the absorbance A was read at a wavelength of 600nm₁And reading the absorbance A when the reaction time is 5 minutes₂Calculating the absorbance change value delta A = A twice₂-A₁. PCT concentration in the sample was calculated by comparison with the same treated calibration solutions. As can be seen from the results of Table 3 and FIG. 3 (FIG. 3 is plotted based on the data of Table 3), the method of the present invention shows very good correlation (correlation R) with the chemiluminescence immunoassay, although the operation is simple and the time required is only within 10 minutes²To 0.9998).

TABLE 3

Example four

One) Procalcitonin reagent 1

Dissolving the raw materials by using 800ml of purified water, supplementing the purified water to a constant volume of 1000ml, and then adjusting the pH value to 7.6 by using hydrochloric acid or sodium hydroxide;

II) Procalcitonin reagent 2

1. Diluting 220nm carboxyl latex microspheres with 50mmol/L, PH 6.0.0 MES buffer solution to obtain 0.01mg/ml suspension;

6. reacting the mixture at 37 ℃ for 3 hours, and continuously stirring;

7. adding ethanolamine into 1ml of the reaction mixture according to the proportion of adding 2.5 mul of ethanolamine, reacting for 20 minutes, and continuously stirring;

8. removing unbound protein and ethanolamine by 15000 r/m.c., diluting with HEPES buffer solution 50mmol/L, PH 7.6.6 to make the concentration of rabbit anti-human PCT antibody latex particles 0.004ml/ml, adding bovine serum albumin (stabilizer) 4mmol/L and sodium azide (preservative) 2mmol/L, dissolving and mixing to obtain procalcitonin reagent 2;

III) liquid type PCT reference calibration substance

And adding 1000ng/ml of the corresponding pure PCT product into the mixed solution according to the required concentration of the PCT reference calibrator to prepare 5 series concentrations of the reference calibrator, wherein the PCT contents of the 5 series concentrations of the reference calibrator are respectively 45ng/ml, 90ng/ml, 180ng/ml, 320ng/ml and 640ng/ml from low to high.

according to the experimental procedure, 240. mu.L of procalcitonin reagent 1 was added to 10. mu.L of the sample, incubated at 37 ℃ for 3 minutes, 60. mu.L of procalcitonin reagent 2 was added, and after incubation at 37 ℃ for 10 seconds, the absorbance A was read at a wavelength of 600nm₁And reading the absorbance A when the reaction time is 5 minutes₂Calculating the absorbance change value delta A = A twice₂-A₁. PCT concentration in the sample was calculated by comparison with the same treated calibration solutions. As can be seen from the results of Table 4 and FIG. 4 (FIG. 4 is plotted based on the data of Table 4), the method of the present invention shows very good correlation (correlation R) with the chemiluminescence immunoassay, although the operation is simple and the time required is only within 10 minutes²To 0.9998).

TABLE 4

The operation steps for determining the procalcitonin reagent and the specific analysis parameters on a full-automatic biochemical analyzer are as follows:

measurement procedure

Analysis parameters (taking parameters of Hitachi 7080 biochemical analyzer as an example)

The method comprises the following steps: two-point end-point method, reaction temperature: 37 deg.C

Dominant wavelength: 520-600nm sub-wavelength: 800nm (optional)

Sample amount: 10 μ L, reagent 1 amount/reagent 2 amount: 240 μ L/60 μ L

The reaction direction is as follows: forward (up), scaling: multipoint nonlinearity

Reading point time: 5 and 21 spots (total reaction time is 420 seconds)

And (3) after the instrument automatically finishes calibration, the instrument can measure the procalcitonin, and the instrument automatically calculates the corresponding PCT concentration.

According to the invention, the PCT antibody is combined with the carboxyl latex microsphere, so that the reaction surface area of the PCT antigen and the antibody is enlarged, and the purpose of measuring a large batch of samples on a conventional biochemical analyzer is realized. Suitable for routine laboratory use in the determination of PCT concentrations. It should be understood that the foregoing is only a preferred embodiment of the present invention and that numerous changes and modifications may be made by those skilled in the art without departing from the principles of the invention and such changes and modifications are to be considered within the scope of the invention.

Claims

1. A reagent for determining procalcitonin comprises the following components in parts by weight:

1) procalcitonin reagent 1

2) procalcitonin reagent 2

(1) Diluting 80nm carboxyl latex microspheres with 50mmol/L MES buffer solution (pH6.0) to obtain 0.01mg/ml suspension;

(2) adding 20mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 40mg of N-hydroxysuccinimide sulfonic acid sodium salt into 1ml of carboxyl latex microsphere suspension, immediately mixing the reagents uniformly, reacting the mixture for 15-30 minutes at room temperature, and continuously stirring;

(3) washing the carboxyl latex microspheres with 50mmol/L MES buffer solution or purified water with pH6.0 to remove unreacted Sulfo-NHS and EDAC, and suspending the latex microspheres in the purified water to obtain a carboxyl latex microsphere suspension, wherein the content of the carboxyl latex microspheres is 0.01 mg/ml;

(4) dissolving goat anti-human procalcitonin antibody in 50mmol/L borate buffer solution with pH of 8.5 to make protein concentration of goat anti-human procalcitonin antibody 0.25 mg/ml;

(5) taking 1ml of the carboxyl latex microsphere suspension in the step (3), and immediately adding 1ml of the goat anti-human procalcitonin antibody in the step (4); the concentrations of the carboxyl latex microspheres, the goat anti-human procalcitonin antibody and the buffer solution in the mixed solution are 0.005mg/ml, 0.125mg/ml and 25mmol/L respectively;

(6) reacting the mixture at 37 ℃ for 3 hours, and continuously stirring;

(7) adding ethanolamine into 1ml of the reaction mixture according to the proportion of adding 2.5 mul of ethanolamine, reacting for 15 minutes, and continuously stirring;

(8) removing unbound protein and ethanolamine by 15000 r/m separation, diluting with 50mmol/L MOPSO buffer solution with pH7.2 to make the concentration of goat anti-human PCT antibody latex particles 0.01ml/ml, adding bovine serum albumin 3mmol/L and 3mmol/L sodium azide, dissolving and mixing well to obtain procalcitonin reagent 2;

3) liquid type PCT reference calibrator

2. A reagent for determining procalcitonin comprises the following components in parts by weight:

1) procalcitonin reagent 1

2) procalcitonin reagent 2

(1) Diluting 150nm carboxyl latex microspheres with 50mmol/L MES buffer solution (pH6.0) to obtain 0.01mg/ml suspension;

(2) according to the proportion of adding 20mg EDAC and 40mg Sulfo-NHS into 1ml carboxyl latex microsphere suspension, uniformly mixing the reagents immediately, reacting the mixture for 15-30 minutes at room temperature, and continuously stirring;

(4) dissolving rabbit anti-human procalcitonin antibody in 50mmol/L borate buffer solution with pH of 8.5 to make the protein concentration of the rabbit anti-human procalcitonin antibody be 0.25 mg/ml;

(5) taking 1ml of the carboxyl latex microsphere suspension in the step (3), and immediately adding 1ml of the rabbit anti-human procalcitonin antibody in the step (4); the concentrations of the carboxyl latex microspheres, the rabbit anti-human procalcitonin antibody and the buffer solution in the mixed solution are 0.005mg/ml, 0.125mg/ml and 25mmol/L respectively;

(6) reacting the mixture at 37 ℃ for 3 hours, and continuously stirring;

(7) adding ethanolamine into 1ml of the reaction mixture according to the proportion of adding 2.5 mul of ethanolamine, reacting for 30 minutes, and continuously stirring;

(8) removing unbound protein and ethanolamine by 15000 r/m separation, diluting with 50mmol/L phosphate buffer solution with pH7.4 to make the concentration of rabbit anti-human PCT antibody latex particles 0.006ml/ml, adding bovine serum albumin 3mmol/L and 3mmol/L sodium azide, dissolving and mixing to obtain procalcitonin reagent 2;

3) liquid type PCT reference calibrator

3. A reagent for determining procalcitonin comprises the following components in parts by weight:

1) procalcitonin reagent 1

2) procalcitonin reagent 2

(1) Diluting 300nm carboxyl latex microspheres with 50mmol/L MES buffer solution (pH6.0) to obtain 0.01mg/ml suspension;

(2) according to the proportion of adding 20mg EDAC and 40mg Sulfo-NHS into 1ml carboxyl latex microsphere suspension, evenly mixing the reagents immediately, reacting the mixture for 15-30 minutes at room temperature, and continuously stirring;

(4) dissolving the mouse anti-human procalcitonin antibody in 50mmol/L borate buffer solution with pH of 8.5 to make the protein concentration of the mouse anti-human procalcitonin antibody be 0.25 mg/ml;

(5) taking 1ml of the carboxyl latex microsphere suspension in the step (3), and immediately adding 1ml of the mouse anti-human procalcitonin antibody in the step (4); the concentrations of the carboxyl latex microspheres, the mouse anti-human procalcitonin antibody and the buffer solution in the mixed solution are 0.005mg/ml, 0.125mg/ml and 25mmol/L respectively;

(6) reacting the mixture at 37 ℃ for 3 hours, and continuously stirring;

(7) adding ethanolamine into 1ml of the reaction mixture according to the proportion of adding 2.5 mul of ethanolamine, reacting for 10 minutes, and continuously stirring;

(8) removing unbound protein and ethanolamine by 15000 r/m separation, diluting with PIPES buffer solution with pH8.0 and 50mmol/L to make the concentration of mouse anti-human PCT antibody latex particles 0.002ml/ml, adding bovine serum albumin 3mmol/L and 3mmol/L sodium azide, dissolving and mixing to obtain procalcitonin reagent 2;

3) liquid type PCT reference calibrator

4. A reagent for determining procalcitonin comprises the following components in parts by weight:

1) procalcitonin reagent 1

2) procalcitonin reagent 2

(1) Diluting 220nm carboxyl latex microspheres with 50mmol/L MES buffer solution (pH6.0) to obtain 0.01mg/ml suspension;

(6) reacting the mixture at 37 ℃ for 3 hours, and continuously stirring;

(7) adding ethanolamine into 1ml of the reaction mixture according to the proportion of adding 2.5 mul of ethanolamine, reacting for 20 minutes, and continuously stirring;

(8) removing unbound protein and ethanolamine by 15000 r/m separation, diluting with 50mmol/L HEPES buffer solution with pH7.6 to make the concentration of rabbit anti-human PCT antibody latex particles 0.004ml/ml, adding bovine serum albumin 4mmol/L and 2mmol/L sodium azide, dissolving and mixing to obtain procalcitonin reagent 2;

3) liquid type PCT reference calibrator