Procalcitonin latex immunoturbidimetry detection reagent combination and application thereof

Abstract

The present application relates to the field of medical immunodetection, in particular to procalcitonin latex immunoturbidimetry detection reagent combination and application thereof. The present application provides procalcitonin latex immunoturbidimetry detection reagent combination comprising R1 reagent and R2 reagent and application thereof. The present application adopts latex immunoturbidimetry, and further provides procalcitonin latex immunoturbidimetry detection reagent combination with good stability, high precision, high accuracy, high anti-interference and high linear range level by optimizing reaction system and proportion of each component, which is conducive to the popularization and application of the reagent in clinic.

Description

Technical Field

[0001] This invention relates to the field of medical immunoassay, specifically to a reagent combination for the detection of calcitonin progeny latex immunoturbidimetric assay and its application. Background Technology

[0002] Procalcitonin (PCT) is a non-hormonal pro-calcitonin glycoprotein composed of 116 amino acids with a molecular weight of 13 kDa. PCT has a half-life of 25-30 hours and exhibits good stability in vitro. PCT is a protein whose plasma levels rise in severe bacterial, fungal, and parasitic infections, as well as in sepsis and multiple organ failure. PCT levels do not rise in autoimmune diseases, allergic reactions, or viral infections. Localized bacterial infections, mild infections, and chronic inflammation do not cause increases in PCT. It plays a crucial role in bacterial endotoxin induction.

[0003] Numerous studies have found that PCT levels are abnormally elevated in systemic bacterial, fungal, and parasitic infections. The degree of elevation is correlated with the severity of the infection and prognosis, and it has high clinical value in the auxiliary differential diagnosis, prognosis assessment, and efficacy monitoring of systemic bacterial infections and sepsis. Monitoring PCT levels is very useful for tracking the progression of serious, life-threatening infectious diseases and monitoring treatment regimens. Elevated PCT concentrations indicate an ongoing inflammatory response. A decrease in PCT values ​​after adequate use of antibiotics and anti-inflammatory treatments demonstrates the correctness of the treatment plan and a good prognosis; conversely, an increase in PCT levels indicates a change in the treatment plan.

[0004] Numerous clinical studies have demonstrated the high value of PCT in diagnosis and treatment guidance across various medical fields. Compared to currently used diagnostic indicators, PCT provides additional information in differential diagnosis and control of infections and severe inflammation. With the continuous advancement of clinical practice research and the accumulation of clinical data, PCT will become a consensus and be widely applied as a routine indicator for the auxiliary and differential diagnosis of systemic bacterial infections and sepsis.

[0005] For PCT testing, existing methods mainly include quantitative, semi-quantitative, and qualitative methods. Quantitative methods include double-antibody sandwich ELISA, immunoluminescence immunoassay, high-performance liquid chromatography (HPLC), and radioimmunoassay. Radioimmunoassay and HPLC molecular methods have certain limitations in clinical practice, while ELISA (double-antibody sandwich ELISA) and immunoluminescence immunoassay are more commonly used clinically. ELISA has high sensitivity, but its operation is complex and time-consuming, generally providing qualitative or semi-quantitative results. Quantitative results have larger deviations and a narrow linear range, and it is difficult to control sample dilution. Immunochemiluminescence immunoassay has the advantage of high sensitivity, meeting the needs of clinical PCT testing, but it requires specialized equipment, is expensive, and is not easily adopted in hospitals. Furthermore, the standard curve drifts over time (due to the long duration of luminescence, measurements can be taken at different times). Qualitative methods mainly include immunochromatography, which can be used as a qualitative or semi-quantitative method, providing rapid results without requiring specialized equipment. However, it cannot provide quantitative data and has a larger subjective error.

[0006] The procalcitonin latex immunoturbidimetric assay kit is based on the binding of PCT in the sample to specific PCT antibodies coated on latex particles, causing an agglutination reaction. The turbidity of the agglutination reaction is directly proportional to the PCT concentration in the sample. This method is an analytical method that requires no sample pretreatment, has low technical and equipment requirements, and offers higher precision and specificity. Because this method does not require expensive equipment, can be automated, and can simultaneously analyze a large number of samples, it has been widely adopted in clinical practice. However, conventional procalcitonin latex immunoturbidimetric assay kits suffer from poor stability, low accuracy, and limited linearity, thus restricting their widespread clinical application. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a reagent combination for the detection of procalcitonin latex immunoturbidimetric assay and its application. The reagent combination for the detection of procalcitonin latex immunoturbidimetric assay provided by the present invention has good stability, high precision, high accuracy, high anti-interference level and high linear range, which is conducive to the promotion and application of the reagent in clinical practice.

[0008] This invention provides a reagent combination, including reagent R1 and reagent R2;

[0009] The R1 reagent comprises 20–100 mM buffer A, 15–45 g / L stabilizer A, 2–5 g / L protectant A, 1.0–5.0 g / L surfactant A, 1.0–2.0 ml / L preservative A, and 9–25 g / L coagulant;

[0010] The R2 reagent comprises 20–100 mM buffer B, 0.1–1 g / L stabilizer B, 2–50 g / L protectant B, 0.1–5.0 g / L surfactant B, 1.0–2.0 ml / L preservative B, and 1.0–1.5 mg / mL polystyrene latex microsphere-calcitonin antibody complex.

[0011] Buffer A includes at least one of MES, Bis-Tris, bisTris-propane, ACES, TES, and HEPES, and buffer B includes at least one of bisTris-propane, ACES, TES, HEPES, TAPS, HEPPS, glycine, and POPSO.

[0012] The stabilizers A and B are independently selected from at least one of sodium chloride, methoxy polyethylene glycol amine 2000 and methoxy polyethylene glycol amine 5000;

[0013] The protective agents A and B are independently selected from at least one of bovine serum albumin, casein, sucrose, trehalose and casein;

[0014] The surfactants A and B are independently selected from at least one of CHAPS, poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), and nonylphenol polyoxyethylene ether, wherein the poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) includes at least one of poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) 12600, poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) 14600, and poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) 1900, and the nonylphenol polyoxyethylene ether includes Tergitol NP-40 and / or Type NP-9;

[0015] The preservatives A and B are independently selected from Proclin 300 and / or NaN3;

[0016] The coagulant includes at least one of PEG6000, PEG8000 and PEG20000.

[0017] Compared with other reagent combinations, the reagent combination provided by this invention uses several buffer solutions and multiple surfactants in reagent R1, which can effectively improve the accuracy of the reagent. Reagent R2 uses stabilizers and surfactants, which can effectively improve the long-term stability of the reagent. At the same time, due to the close cooperation between the components of the reagent combination, the stability, precision, accuracy, anti-interference ability and linear range of the reagent combination of this invention are further guaranteed and improved, thereby achieving better technical results.

[0018] In some embodiments, buffer A is a bisTris-propane buffer and buffer B is a TAPS buffer.

[0019] In some embodiments, stabilizer A is sodium chloride, and stabilizer B is methoxy polyethylene glycol amine 5000.

[0020] In some embodiments, the protective agent A is casein, and the protective agent B is trehalose and casein.

[0021] In some embodiments, surfactant A is CHAPS and Type NP-9, and surfactant B is CHAPS.

[0022] In some embodiments, preservative A and preservative B are Proclin 300.

[0023] In some embodiments, the coagulant is PEG8000.

[0024] In some embodiments, the polystyrene latex microspheres are composed of microspheres with a wavelength of 200 nm to 650 nm.

[0025] In some specific embodiments, the R1 reagent comprises 12.52 g / L bisTris-propane buffer, 39 g / L NaCl, 2 g / L casein, 2 g / L CHAPS, 1 g / L Type NP-9; 1.0 mL / L Proclin 300 and 15 g / L PEG8000;

[0026] The R2 reagent comprises 20 mM TAPS buffer, 0.5 g / L methoxy polyethylene glycolamine 5000, 20 g / L casein, 20 g / L trehalose, 1 g / L CHAPS, 1 mL / L Proclin 300, and 1.25 mg / mL polystyrene latex microspheres-procalcitonin antibody complex.

[0027] In some embodiments, the preparation method of the R2 reagent includes: taking activated carboxylated polystyrene latex microspheres and reacting them with two procalcitonin antibodies to obtain the polystyrene latex microsphere-procalcitonin antibody complex, then mixing it with the buffer B, stabilizer B, protectant B, surfactant B and preservative B, and heat stabilizing it for 45-60 hours.

[0028] In some specific embodiments, the carboxylated polystyrene latex microspheres have a particle size of 500 nm, the activation reaction uses EDC reagent, the reaction is terminated by bovine serum albumin after mixing, and the heat stabilization treatment is performed at 45°C for 60 h.

[0029] This invention provides the application of the aforementioned reagent combination in the preparation of a procalcitonin detection kit.

[0030] This invention provides a procalcitonin detection kit, comprising the aforementioned reagent combination.

[0031] This invention provides a method for detecting procalcitonin, comprising: detecting a sample using the reagent combination or procalcitonin detection kit described in this invention.

[0032] This invention provides a reagent combination for the detection of procalcitonin using latex immunoturbidimetric assay, comprising reagents R1 and R2, and its application. This invention employs latex immunoturbidimetry and, through optimization of the reaction system and the ratio of each component, further provides a reagent combination for the detection of procalcitonin using latex immunoturbidimetric assay with good stability, high precision, accuracy, anti-interference, and high linear range, which is beneficial for the widespread clinical application of the reagent. Compared with existing procalcitonin detection kits, the reagent combination provided by this invention uses surfactants CHAPS and nonylphenol polyoxyethylene ether to enhance the reagent's anti-interference ability. Reagent R1 uses several buffer solutions and multiple surfactants, optimizing the ratio of each component to improve reagent accuracy and precision; reagent R2 uses a special stabilizer (methoxy polyethylene glycol amine 2000 / 5000), surfactant, and heat stabilization treatment to significantly improve the long-term stability of the reagent. Attached Figure Description

[0033] Figure 1 The correlation diagram between the detection results of the PCT detection kit and the control kit in Example 1 of this invention is shown.

[0034] Figure 2 The graph shows the linear relationship between the detected PCT concentration value and the theoretical value of the kit in Example 1 of this invention. Detailed Implementation

[0035] This invention provides a reagent combination for the detection of calcitonin progeny latex immunoturbidimetric assay and its application. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0036] A calcitonin progeny latex immunoturbidimetric assay kit includes:

[0037] Standards; the concentrations of the standards are 50, 30, 10, 2, 0.5, and 0 ng / mL, respectively.

[0038] R1 reagent; R1 components include: buffer A, stabilizer A, protectant A, sensitizer, surfactant A, and preservative A.

[0039] R2 reagent; R2 components include: buffer B, stabilizer B, protectant B, surfactant B, preservative B, and polystyrene latex microsphere-calcitonin antibody complex.

[0040] The test materials used in this invention are all commercially available products, and are readily available in the market.

[0041] Antibodies A and B were purchased from Oukai Biotechnology, with catalog numbers K3b8 and K4c9, respectively.

[0042] The present invention will be further illustrated below with reference to the embodiments.

[0043] Example 1: The reagent kit of the present invention

[0044] 1) Preparation of reagent R1:

[0045] Weigh 12.52g of bisTris propane, 39g of NaCl, 2g of casein, 15g of PEG8000, 2.0g of CHAPS, 1.0g of Type NP-9, and 1.0ml of Proclin300 into a clean container. Add deionized water and stir for 30 minutes to fully dissolve the contents. Adjust the pH to 6.8. Add deionized water to a final volume of 1L and filter to obtain R1.

[0046] 2) Preparation of reagent R2:

[0047] Step 1: Add 500 nm diameter carboxylated polystyrene latex microspheres to 10 mmol / L LMES buffer to make the concentration 8.3 mg / ml;

[0048] Step 2: Add 4 mg / ml EDC to the above latex microspheres and stir at room temperature for 0.5 h;

[0049] Step 3: Dissolve and dilute PCT antibodies A and B to 0.27 mg / ml with citrate buffer at pH 4.5. Then add the diluted antibody A and B solutions to the activated polystyrene latex microspheres and stir at room temperature for 3 hours.

[0050] Step 4: Add 10% bovine serum albumin to terminate the reaction, stir at room temperature for 0.5 h; centrifuge the resulting reaction solution, discard the supernatant, and obtain the precipitate;

[0051] Step 5: Disperse the precipitate with 20 mmol / L TAPS buffer, then add 0.5 g methoxy polyethylene glycol 5000; 20 g casein; 20 g trehalose; 1.0 g CHAPS; and 1.0 ml Proclin 300 and stir well. Mix the two latex groups evenly to obtain R2, with a pH of 7.5 and a total volume of 1 L. The concentration of the polystyrene latex microsphere-calcitonin antibody complex is 1.25 mg / mL.

[0052] Step 6: Place the above R2 in a 45-degree water bath for 60 hours to achieve heat stabilization and obtain the final R2 reagent.

[0053] Example 2: The reagent kit of the present invention

[0054] 1) Preparation of reagent R1:

[0055] Weigh 12.52g of bisTris propane, 39g of NaCl, 2g of BSA, 15g of PEG8000, 2.0g of 12600, and 1.0ml of Proclin300 into a clean container according to the formula. Add deionized water and stir for 30 minutes to fully dissolve the contents. Adjust the pH to 6.8. Add deionized water to a final volume of 1L and filter to obtain R1.

[0056] 2) Preparation of reagent R2:

[0057] Step 1: Add 500 nm diameter carboxylated polystyrene latex microspheres to 10 mmol / L LMES buffer to make the concentration 8.3 mg / ml;

[0058] Step 2: Add 4 mg / ml EDC to the above latex microspheres and stir at room temperature for 0.5 h;

[0059] Step 3: Dissolve and dilute PCT antibodies A and B to 0.27 mg / ml with citrate buffer at pH 4.5. Then add the diluted antibody A and B solutions to the activated polystyrene latex microspheres and stir at room temperature for 3 hours.

[0060] Step 4: Add 10% bovine serum albumin to terminate the reaction, stir at room temperature for 0.5 h; centrifuge the resulting reaction solution, discard the supernatant, and obtain the precipitate;

[0061] Step 5: Disperse the precipitate with 20 mmol / L TAPS buffer, then add 0.5 g methoxy polyethylene glycol amine 2000; 20 g casein; 20 g trehalose; 1.0 g CHAPS; and 1.0 ml Proclin 300 and stir well. Mix the two latex groups evenly to obtain R2, with a pH of 7.5 and a total volume of 1 L. The concentration of the polystyrene latex microsphere-calcitonin antibody complex is 1.25 mg / mL.

[0062] Step 6: Place the above R2 in a 45-degree water bath for 55 hours to achieve heat stabilization and obtain the final R2 reagent.

[0063] Example 3: The reagent kit of the present invention

[0064] 1) Preparation of reagent R1:

[0065] Weigh out 10.66g MES, 39g NaCl, 1g BSA, 1g Casein, 9g PEG20000, 1.0g 12600, 1.0g CHAPS, and 1.0ml Proclin300 into a clean container according to the formula. Add deionized water and stir for 30 minutes to fully dissolve the ingredients. Adjust the pH to 5.4. Add deionized water to a final volume of 1L and filter to obtain R1.

[0066] 2) Preparation of reagent R2:

[0067] Step 1: Add 500 nm diameter carboxylated polystyrene latex microspheres to 10 mmol / L LMES buffer to make the concentration 8.3 mg / ml;

[0068] Step 2: Add 4 mg / ml EDC to the above latex microspheres and stir at room temperature for 0.5 h;

[0069] Step 3: Dissolve and dilute PCT antibodies A and B to 0.27 mg / ml with citrate buffer at pH 4.5. Then add the diluted antibody A and B solutions to the activated polystyrene latex microspheres and stir at room temperature for 3 hours.

[0070] Step 4: Add 10% bovine serum albumin to terminate the reaction, stir at room temperature for 0.5 h; centrifuge the resulting reaction solution, discard the supernatant, and obtain the precipitate;

[0071] Step 5: Disperse the precipitate with 20 mmol / L TAPS buffer, then add 0.5 g methoxy polyethylene glycol 2000; 0.5 g methoxy polyethylene glycol 5000; 20 g casein; 20 g trehalose; 2.0 g CHAPS; and 1.0 ml Proclin 300 and stir well. Mix the two latex groups evenly to obtain R2, with a pH of 8.0 and a total volume of 1 L. The concentration of the polystyrene latex microsphere-calcitonin antibody complex is 1.25 mg / mL.

[0072] Step 6: Place the above R2 in a 45-degree water bath for 45 hours to achieve heat stabilization and obtain the final R2 reagent.

[0073] Comparative Example 1

[0074] A PCT detection kit, comprising reagent R1 and reagent R2, is identical to that in Example 1, except that reagent R1 comprises the following components: 12.52g bisTris propane; 39g NaCl; 2g Casein; 15g PEG8000; 1.0ml Proclin 300.

[0075] The preparation method of the R1 reagent includes:

[0076] Weigh out the required amounts of bisTris propane buffer, sodium chloride, Casein, polyethylene glycol 8000, and preservative Proclin 300, put them into a stirrer and stir well, then adjust the pH value; add deionized water to 1L, stir well, and filter to obtain R1.

[0077] Comparative Example 2

[0078] A PCT detection kit, comprising reagent R1 and reagent R2, is consistent with Example 1, except that: reagent R1 comprises the following components: 10.463g MOPS; 39g NaCl; 2g casein; 15g PEG8000; 2.0g CHAPS; 1.0g Type NP-9; 1.0ml Proclin300;

[0079] The preparation method of the R1 reagent includes:

[0080] Weigh out the required amounts of MOPS buffer, sodium chloride, Casein, polyethylene glycol 8000, surfactant CHAPS, Type NP-9, and preservative Proclin 300, put them into a stirrer and stir well, then adjust the pH value; add deionized water to 1L, stir well, and filter to obtain R1.

[0081] Comparative Example 3

[0082] A PCT detection kit, comprising reagent R1 and reagent R2, is consistent with Example 1, except that: reagent R1 comprises the following components: 7.098g phosphate; 39g NaCl; 2g casein; 15g PEG8000; 2.0g CHAPS; 1.0g Type NP-9; 1.0ml Proclin300;

[0083] The preparation method of the R1 reagent includes:

[0084] Weigh out the required amounts of phosphate buffer, sodium chloride, Casein, polyethylene glycol 8000, surfactant CHAPS, Type NP-9, and preservative Proclin 300, put them into a stirrer and stir well, then adjust the pH value; add deionized water to 1L, stir well, and filter to obtain R1.

[0085] Comparative Example 4

[0086] A PCT detection kit includes reagent R1 and reagent R2, which are the same as in Example 1, except that: the R2 reagent does not contain the stabilizer methoxy polyethylene glycol 5000 or the surfactant CHAPS during preparation, and is heat stabilized at 45 degrees Celsius for 15 hours.

[0087] The preparation method of the R2 reagent:

[0088] Step 1: Add 500 nm diameter carboxylated polystyrene latex microspheres to 10 mmol / L LMES buffer to make the concentration 8.3 mg / ml;

[0089] Step 2: Add 4 mg / ml EDC to the above latex microspheres and stir at room temperature for 0.5 h;

[0090] Step 3: Dissolve and dilute PCT antibodies A and B to 0.27 mg / ml with citrate buffer at pH 4.5. Then add the diluted antibody A and B solutions to the activated polystyrene latex microspheres and stir at room temperature for 3 hours.

[0091] Step 4: Add 10% bovine serum albumin to terminate the reaction, stir at room temperature for 0.5 h; centrifuge the resulting reaction solution, discard the supernatant, and obtain the precipitate;

[0092] Step 5: Disperse the precipitate with 20 mmol / L TAPS buffer, then add 20 g casein, 20 g trehalose, and 1.0 ml Proclin 300 and stir well. Mix the two groups of latex evenly to obtain R2, with a pH of 7.5 and a total volume of 1 L. The concentration of the polystyrene latex microsphere-calcitonin antibody complex is 1.25 mg / mL.

[0093] Step 6: Place the above R2 in a 45-degree water bath for 15 hours to achieve heat stabilization and obtain the final R2 reagent.

[0094] Comparative Example 5

[0095] A PCT detection kit includes reagent R1 and reagent R2, which is consistent with Example 1, except that the stabilizer methoxy polyethylene glycol 5000 is not added during the preparation of reagent R2.

[0096] The preparation method of the R2 reagent:

[0097] Step 1: Add 500 nm diameter carboxylated polystyrene latex microspheres to 10 mmol / L LMES buffer to make the concentration 8.3 mg / ml;

[0098] Step 2: Add 4 mg / ml EDC to the above latex microspheres and stir at room temperature for 0.5 h;

[0099] Step 3: Dissolve and dilute PCT antibodies A and B to 0.27 mg / ml with citrate buffer at pH 4.5. Then add the diluted antibody A and B solutions to the activated polystyrene latex microspheres and stir at room temperature for 3 hours.

[0100] Step 4: Add 10% bovine serum albumin to terminate the reaction, stir at room temperature for 0.5 h; centrifuge the resulting reaction solution, discard the supernatant, and obtain the precipitate;

[0101] Step 5: Disperse the precipitate with 20 mmol / L TAPS buffer, then add 20 g casein, 20 g trehalose, 1.0 g CHAPS, and 1.0 ml Proclin 300 and stir well. Mix the two latex groups evenly to obtain R2, with a pH of 7.5 and a total volume of 1 L. The concentration of the polystyrene latex microsphere-calcitonin antibody complex is 1.25 mg / mL.

[0102] Step 6: Place the above R2 in a 45-degree water bath for 60 hours to achieve heat stabilization and obtain the final R2 reagent.

[0103] Comparative Example 6

[0104] A PCT detection kit includes reagent R1 and reagent R2, which is consistent with Example 1, except that the surfactant CHAPS is not added during the preparation of reagent R2.

[0105] The preparation method of the R2 reagent:

[0106] Step 1: Add 500 nm diameter carboxylated polystyrene latex microspheres to 10 mmol / L LMES buffer to make the concentration 8.3 mg / ml;

[0107] Step 2: Add 4 mg / ml EDC to the above latex microspheres and stir at room temperature for 0.5 h;

[0108] Step 3: Dissolve and dilute PCT antibodies A and B to 0.27 mg / ml with citrate buffer at pH 4.5. Then add the diluted antibody A and B solutions to the activated polystyrene latex microspheres and stir at room temperature for 3 hours.

[0109] Step 4: Add 10% bovine serum albumin to terminate the reaction, stir at room temperature for 0.5 h; centrifuge the resulting reaction solution, discard the supernatant, and obtain the precipitate;

[0110] Step 5: Disperse the precipitate with 20 mmol / L TAPS buffer, then add 0.5 g methoxy polyethylene glycol amine 5000; 20 g casein; 20 g trehalose; and 1.0 ml Proclin 300 and stir well. Mix the two groups of latexes evenly to obtain R2, with a pH of 7.5 and a total volume of 1 L. The concentration of the polystyrene latex microsphere-calcitonin antibody complex is 1.25 mg / mL.

[0111] Step 6: Place the above R2 in a 45-degree water bath for 60 hours to achieve heat stabilization and obtain the final R2 reagent.

[0112] Comparative Example 7

[0113] A PCT detection kit includes reagent R1 and reagent R2, which is consistent with Example 1, except that reagent R2 is heat-stabilized at 45 degrees Celsius for 15 hours during preparation.

[0114] The preparation method of the R2 reagent:

[0115] Step 1: Add 500 nm diameter carboxylated polystyrene latex microspheres to 10 mmol / L LMES buffer to make the concentration 8.3 mg / ml;

[0116] Step 2: Add 4 mg / ml EDC to the above latex microspheres and stir at room temperature for 0.5 h;

[0117] Step 3: Dissolve and dilute PCT antibodies A and B to 0.27 mg / ml with citrate buffer at pH 4.5. Then add the diluted antibody A and B solutions to the activated polystyrene latex microspheres and stir at room temperature for 3 hours.

[0118] Step 4: Add 10% bovine serum albumin to terminate the reaction, stir at room temperature for 0.5 h; centrifuge the resulting reaction solution, discard the supernatant, and obtain the precipitate;

[0119] Step 5: Disperse the precipitate with 20 mmol / L TAPS buffer, then add 0.5 g methoxy polyethylene glycol 5000; 20 g casein; 20 g trehalose; 1.0 g CHAPS; and 1.0 ml Proclin 300 and stir well. Mix the two latex groups evenly to obtain R2, with a pH of 7.5 and a total volume of 1 L. The concentration of the polystyrene latex microsphere-calcitonin antibody complex is 1.25 mg / mL.

[0120] Step 6: Place the above R2 in a 45-degree water bath for 15 hours to achieve heat stabilization and obtain the final R2 reagent.

[0121] Example of effect

[0122] The performance evaluation results of the reagent kits described in Examples 1-3 of this invention are basically consistent. Taking the reagent kit prepared in Example 1 as an example, the correlation, linear range, precision, anti-interference, stability and other related performances are verified.

[0123] (1) Standard curve development

[0124] The reagents used in the above implementation method were tested using a Toshiba 120FR fully automated biochemical analyzer at a wavelength of 700 nm. 15 μL of sample or calibrator was taken, and 135 μL of reagent R1 was added. The mixture was kept at 37°C for 5 min, followed by the addition of 45 μL of reagent R2. After 20 s, the absorbance A1 was read. After incubation at 37°C for 5 min, the absorbance A2 was read. The reaction absorbance ΔA = A2 - A1. First, multi-point calibration was performed using standards, and the calibration curve was calculated using spline functions. The procalcitonin concentration in the sample could be found from the working curve.

[0125] (2) Correlation experiment:

[0126] A commercially recognized procalcitonin kit with excellent accuracy was used as the control group, while the kit from Example 1 was used as the experimental group for comparison. Fifty samples were tested, and the results are shown in Table 1. When testing low-value samples, the probability of a 0 value was lower than that of the control kit, thus demonstrating higher analytical sensitivity.

[0127] Using the test results of commercially available control kits as the independent variable on the x-axis and the test results of the kit of this invention as the dependent variable on the y-axis, a linear regression curve was plotted. The regression equation for Example 1 was Y = 0.9823X + 0.0646, with a linear correlation coefficient R = 0.9988. The linear relationship was good, and the test results can be effectively used for clinical testing. The correlation curve for Example 1 is shown below. Figure 1 .

[0128] The experimental results of Comparative Examples 1-3 are shown in Table 1; the regression equation for Comparative Example 1 is Y = 0.8207X + 1.3948, with a linear correlation coefficient R = 0.9552; the regression equation for Comparative Example 2 is Y = 0.8275X + 1.0579, with a linear correlation coefficient R = 0.9694; the regression equation for Comparative Example 3 is Y = 0.8687X + 0.8456, with a linear correlation coefficient R = 0.9744.

[0129] The results in Table 1 show that using special buffer solutions and surfactants in the R1 reagent can significantly improve the accuracy of the reagent.

[0130] Table 1. Results of correlation experiments for Example 1 and Comparative Examples 1-3 (Unit: ng / mL)

[0131]

[0132]

[0133] (3) Linear experiment:

[0134] Eleven dilutions were prepared by mixing high and low value samples (arithmically equal), and each sample was measured twice and the mean was taken. Normal samples were mixed to obtain a low-value sample of 0.05 ng / mL. A high-value PCT sample of 50 ng / mL was added to the mixed normal samples as the linear high value. The high and low value samples were then mixed proportionally to form eleven dilutions. The linear determination results are shown in Table 2. A linear regression equation was derived using the theoretical PCT concentration as the independent variable X on the x-axis and the actual measured value as the dependent variable Y on the y-axis. The correlation coefficient r was calculated, and the results showed that the linear regression equation was Y = 1.0098X + 0.0844, with a correlation coefficient r = 0.9998, indicating that the present invention exhibits good correlation within the linear range of 0.05 ng / mL to 50 ng / mL.

[0135] Table 2 Results of Linear Range Analysis

[0136]

[0137] Based on the data in Table 2, construct a linear range curve, such as... Figure 2 .

[0138] (4) Precision testing

[0139] Serum samples with high, medium, and low PCT concentrations were taken and tested 20 times consecutively. The coefficient of variation (CV) was calculated. The precision results of Example 1 and Comparative Examples 1-3 are shown in Table 3. The reagent of Example 1 of this invention demonstrated good precision in determining high, medium, and low concentration serum samples, with CV values ​​all below 5%. Table 3 shows that using the R1 buffer type mentioned in this invention and adding a surfactant to R1 significantly improves reagent precision; changing the R1 buffer type or not adding a surfactant to R1 results in poorer reagent precision.

[0140] Table 3 Precision test results

[0141]

[0142] (5) Anti-interference detection

[0143] Serum from clinically healthy patients was divided into two portions. One portion was treated with the highest concentration of the interfering substance, while the other portion was treated with an equal volume of solvent. The samples with and without the interfering substance were then subjected to three graded dilutions. Each sample was tested three times, with the testing order reversed.

[0144] The deviation of the measured values ​​was calculated. The anti-interference ability of Example 1 and Comparative Example 1 was evaluated, and the results are shown in Table 4.

[0145] The results in Table 4 show that adding surfactants CHAPS and Type NP-9 to R1 can significantly improve the reagent's ability to resist hemoglobin and fat emulsion interference.

[0146] Table 4. Anti-interference test results of Example 1 and Comparative Example 1

[0147]

[0148] (6) Stability test

[0149] Long-term stability tests were conducted on the reagent kit of this invention. The kit was calibrated on a testing instrument and stored in a sealed container at 2–8°C for 18 months. Long-term stability tests were performed on serum samples with concentrations of 1.10 ng / mL and 5.05 ng / mL at 3, 6, 9, 12, and 18 months, respectively. The deviation values ​​of the test results after 18 months of opening were calculated, and the results are shown in Table 5. Table 5 shows that: Comparative Examples 5-7 demonstrate that the combined use of the stabilizer methoxy polyethylene glycol amine and the surfactant CHAPS significantly improves the long-term stability of the reagent; Comparative Example 7 shows that, based on the combined use of the stabilizer methoxy polyethylene glycol amine and the surfactant CHAPS, heat stabilization treatment of the reagent has a significant effect on improving reagent stability. The combined use of these three conditions can significantly improve reagent stability.

[0150] Table 5. Stability test results of Example 1 and Comparative Examples 4-7

[0151]

[0152] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A reagent combination, characterized in that The R1 reagent and the R2 reagent; The R1 reagent comprises 20-100 mM double Tris-propane buffer, 15-45 g / L sodium chloride, 2-5 g / L casein, 1.0-5.0 g / L surfactant A, 1.0-2.0 ml / L Proclin 300 and 9-25 g / L PEG 8000; The R2 reagent comprises 20-100 mM TAPS buffer, 0.1-1 g / L methoxy polyethylene glycol amine 5000, 2-50 g / L protective agent B, 0.1-5.0 g / L CHAPS, 1.0-2.0 ml / L Proclin 300 and 1.0-1.5 mg / mL polystyrene latex microspheres-calcitonin precursor antibody complex; The protective agent B is composed of trehalose and casein in a mass ratio of 1:1, and the surfactant A is composed of CHAPS and Type NP-9 in a mass ratio of 2:

1.

2. The combination of agents according to claim 1, characterized in that, The preparation method of the R2 reagent comprises: after the activated carboxylated polystyrene latex microspheres are mixed and reacted with two strains of calcitonin precursor antibodies to obtain the polystyrene latex microspheres-calcitonin precursor antibody complex, the polystyrene latex microspheres-calcitonin precursor antibody complex is mixed with TAPS buffer, methoxy polyethylene glycol amine 5000, protective agent B, CHAPS and Proclin 300, and is subjected to heat stabilization treatment for 45-60 h.

3. Use of the reagent combination of claim 1 or 2 in the preparation of a calcitonin precursor detection kit.

4. A procalcitonin test kit, characterized in that The reagent combination of claim 1 or 2. The reagent combination of claim 1 or 2.

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