Diluent for peroxidase conjugates and use thereof

Abstract

The application provides a diluent of a peroxidase conjugate and application thereof, and the diluent comprises a basic buffer, a coagulant, a surfactant, an alkali metal chloride and an inert protein; the surfactant is selected from one or more of polyoxyethylene fatty alcohol ether-1, polyoxyethylene fatty alcohol ether-4 and polyoxyethylene fatty alcohol ether-5; the concentration of the coagulant is 0.02w / v% to 0.1w / v%; the concentration of the surfactant is 0.02w / v% to 0.5w / v%; the concentration of the alkali metal chloride is 0.5w / v% to 1w / v%; and the concentration of the inert protein is 0.1w / v% to 0.5w / v%. The diluent can reduce the background signal value of chemiluminescence immunoassay, and improve the signal-to-noise ratio of each detection index, so as to improve the sensitivity and accuracy of the chemiluminescence immunoassay technology.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a diluent for a peroxidase conjugate and its application. Background Technology

[0002] Chemiluminescence immunoassay (CLIA) is a quantitative immunoassay technique that combines chemiluminescence and specific reactions between antigens and antibodies. Due to its advantages such as wide linear range, high sensitivity, and strong specificity, it has become the mainstream method in immunoassay techniques. The high sensitivity and accuracy of immunoassay detection are based on a good signal-to-noise ratio (the ratio of the signal value of the specifically bound target analyte to the background signal value generated by non-specific adsorption).

[0003] In chemiluminescence immunoassay, the most widely used method is to conjugate horseradish peroxidase with antibodies to form peroxidase conjugates, followed by enzymatic chemiluminescence. Peroxidase conjugates are typically prepared as high-concentration stock solutions and then diluted to ready-to-use working concentrations (1:10 to 1:100). However, traditional peroxidase conjugate diluents often result in excessively high background values ​​and low signal-to-noise ratios in the chemiluminescence signal. Therefore, improving the signal-to-noise ratio, and consequently enhancing detection sensitivity and accuracy, while maintaining enzyme activity and protein stability remains a significant challenge. Summary of the Invention

[0004] Based on this, this application provides a diluent for peroxidase conjugates that can maintain sample stability and improve the signal-to-noise ratio.

[0005] In addition, an application of the diluent for the above-mentioned peroxidase conjugate is also provided.

[0006] According to one aspect of this application, a diluent for a peroxidase conjugate is provided, comprising a basal buffer, a coagulant, a surfactant, an alkali metal chloride, and an inert protein; wherein the surfactant is selected from one or more of polyoxyethylene fatty alcohol ether-1, polyoxyethylene fatty alcohol ether-4, and polyoxyethylene fatty alcohol ether-5; the concentration of the coagulant is 0.02 w / v% to 0.1 w / v%; the concentration of the surfactant is 0.02 w / v% to 0.5 w / v%; the concentration of the alkali metal chloride is 0.5 w / v% to 1 w / v%; and the concentration of the inert protein is 0.1 w / v% to 0.5 w / v.

[0007] In one embodiment, the concentration of the surfactant is 0.03 w / v% to 0.08 w / v%; and / or,

[0008] The concentration of the solute in the basal buffer solution is 25 mmol / L to 50 mmol / L.

[0009] In one embodiment, the pH value of the diluent is 6.0 to 7.0.

[0010] In one embodiment, the base buffer is selected from one or more of the following buffers: HEPES buffer, MOPS buffer, and PBS buffer.

[0011] In one embodiment, the coagulant is selected from one or more of polyethylene glycol 1000, polyethylene glycol 2000, polyethylene glycol 6000 and polyethylene glycol 8000.

[0012] In one embodiment, the surfactant is selected from one or more of polyoxyethylene fatty alcohol ether-1, polyoxyethylene fatty alcohol ether-4, and polyoxyethylene fatty alcohol ether-5.

[0013] In one embodiment, the alkali metal chloride is selected from one or more of sodium chloride and calcium chloride.

[0014] In one embodiment, the inert protein is selected from one or more of human serum albumin, bovine serum albumin, and horse serum albumin.

[0015] In one embodiment, the diluent further includes a preservative selected from one or more of sodium azide and ProClin300.

[0016] According to another aspect of this application, a chemiluminescent immunoassay method is provided, in which the peroxidase conjugate is diluted with the diluent of the peroxidase conjugate described above and then subjected to chemiluminescent immunoassay.

[0017] Compared with the prior art, this application has the following beneficial effects:

[0018] The diluent for the peroxidase conjugate in this application can reduce the background signal value in the chemiluminescence immunoassay detection process while maintaining enzyme activity and protein stability, thereby improving the signal-to-noise ratio of each detection index and enhancing the sensitivity and accuracy of chemiluminescence immunoassay technology. Attached Figure Description

[0019] Figure 1 The signal-to-noise ratio of the diluent used in Example 1 for chemiluminescent immunoassay detection;

[0020] Figure 2 The signal-to-noise ratio of different types of surfactants used in chemiluminescent immunoassay detection in Example 4;

[0021] Figure 3 The background signal values ​​for different concentrations of surfactant used in Example 5 for chemiluminescent immunoassay detection are shown. Detailed Implementation

[0022] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, a detailed description of specific embodiments of this application is provided. Many specific details are set forth in the following description to provide a thorough understanding of this application. However, this application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this application. Therefore, this application is not limited to the specific embodiments disclosed below.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Unless otherwise specifically stated, all raw materials, reagents, instruments, and equipment used in this application are commercially available or can be prepared by existing methods.

[0024] "Inert proteins" refer to proteins that do not have biological activity (enzyme activity, antigen-antibody reactions, etc.) in the reaction system, and only play a role in increasing protein concentration and reducing non-specific binding, which is the opposite of the role of active proteins.

[0025] This application provides a diluent for a peroxidase conjugate, comprising a basal buffer, a coagulant, a surfactant, an alkali metal chloride, and an inert protein; wherein the surfactant is selected from one or more of polyoxyethylene fatty alcohol ether-1, polyoxyethylene fatty alcohol ether-4, and polyoxyethylene fatty alcohol ether-5; the concentration of the coagulant is 0.02 w / v% to 0.1 w / v%; the concentration of the surfactant is 0.02 w / v% to 0.5 w / v%; the concentration of the alkali metal chloride is 0.5 w / v% to 1 w / v%; and the concentration of the inert protein is 0.1 w / v% to 0.5 w / v.

[0026] Surfactants can elute non-specific adsorption, reduce hydrophobic interactions between proteins, and improve specific recognition capabilities. In some embodiments, the surfactant is selected from one or more of polyoxyethylene fatty alcohol ether-1, polyoxyethylene fatty alcohol ether-4, and polyoxyethylene fatty alcohol ether-5. Surfactants have the function of refolding antigens, have a strong elution effect on non-specific adsorption, and can also reduce hydrophobic interactions between proteins, thereby improving specific recognition capabilities. The inventors of this application have discovered through research that as the concentration of surfactant increases, the background signal value decreases; however, when the surfactant concentration is too high, the signal value of the target analyte also decreases. Therefore, controlling the concentration of the surfactant to 0.02 w / v% to 0.5 w / v% can effectively reduce the background signal value without affecting the signal value of the target analyte. In some optional specific examples, the surfactant concentration is 0.02 w / v%, 0.05 w / v%, 0.1 w / v%, 0.2 w / v%, or 0.5 w / v.

[0027] In some embodiments, the concentration of the surfactant is 0.02 w / v% to 0.2 w / v%.

[0028] Furthermore, the surfactant concentration was 0.03 w / v% to 0.08 w / v%. Once the surfactant concentration reached 0.03 w / v% to 0.08 w / v%, further increasing the concentration did not result in a significant decrease in the background signal value. Even further, the surfactant concentration was 0.05 w / v%.

[0029] A basal buffer ensures the stability of the diluent during long-term storage, preventing sedimentation; an appropriate amount of coagulant accelerates the antigen-antibody reaction. In some embodiments, the basal buffer is selected from one or more of the following buffers: HEPES buffer, MOPS buffer, and PBS buffer. The basal buffer possesses buffering capacity, high solubility, and good biocompatibility, with its pH value changing minimally with external influences. Adding a basal buffer to the peroxidase conjugate diluent provides a suitable reaction environment (pH, osmotic pressure, ionic strength, etc.) for the subsequent specific binding of antigen and antibody. Furthermore, adding a basal buffer with a solute concentration of 25 mmol / L to 50 mmol / L ensures the stability of the peroxidase conjugate diluent during long-term storage, preventing reagent sedimentation, pH fluctuations, and decreased reaction signal values.

[0030] In some embodiments, the concentration of the solute in the basal buffer is 25 mmol / L to 50 mmol / L.

[0031] In some optional specific examples, the concentration of the solute in the basal buffer is 25 mmol / L, 30 mmol / L, 45 mmol / L, or 50 mmol / L.

[0032] Furthermore, the concentration of the solute in the basal buffer is 35 mmol / L to 50 mmol / L.

[0033] In some embodiments, the diluent for the peroxidase conjugate has a pH of 6.0 to 7.0. In some optional specific examples, the diluent for the peroxidase conjugate has a pH of 6.0, 6.3, 6.5, 6.7, or 7.0. It is understood that a pH of 6.0 to 7.0 allows for better specific binding of the antigen and antibody in the reaction system.

[0034] In some embodiments, the coagulant is selected from one or more of polyethylene glycol 1000, polyethylene glycol 2000, polyethylene glycol 6000, and polyethylene glycol 8000. Adding an appropriate amount of coagulant facilitates a rapid reaction between antigen and antibody, thereby increasing the detection speed.

[0035] In some specific examples, the concentration of the coagulant was 0.02 w / v% to 0.1 w / v%.

[0036] In some optional specific examples, the concentration of the coagulant is 0.02 w / v%, 0.04 w / v%, 0.08 w / v%, or 0.1 w / v.

[0037] Furthermore, the concentration of the coagulant is 0.02 w / v% to 0.05 w / v%.

[0038] Alkali metal chlorides are beneficial for maintaining conductivity, osmotic pressure, and ionic strength, thus maintaining the optimal conditions required for antigen-antibody reactions. In some embodiments, the alkali metal chloride is selected from one or more of sodium chloride and calcium chloride. Adding an appropriate amount of alkali metal chloride to the peroxidase conjugate diluent helps maintain the conductivity, osmotic pressure, and optimal ionic strength of the buffer solution, thereby maintaining the optimal conditions required for antigen-antibody reactions and ensuring the accuracy and reliability of test results.

[0039] In some embodiments, the concentration of the alkali metal chloride is 0.5 w / v% to 1 w / v%.

[0040] In some optional specific examples, the concentration of alkali metal chloride is 0.5 w / v%, 0.6 w / v%, 0.7 w / v%, 0.8 w / v%, or 1 w / v.

[0041] Furthermore, the concentration of alkali metal chlorides is 0.8 w / v% to 1 w / v.

[0042] Inert proteins can increase the concentration of proteins in solution, protect enzymes, and reduce high background levels caused by nonspecific binding. In some embodiments, the inert protein is selected from one or more of human serum albumin, bovine serum albumin, and horse serum albumin.

[0043] In some embodiments, the concentration of the inert protein is 0.1 w / v% to 0.5 w / v%.

[0044] In some optional specific examples, the concentration of the inert protein is 0.1 w / v%, 0.2 w / v%, 0.3 w / v%, 0.4 w / v%, or 0.5 w / v.

[0045] Furthermore, the concentration of the inert protein was 0.1 w / v% to 0.25 w / v%.

[0046] In some embodiments, the diluent further includes a preservative. The preservative effectively inhibits microbial growth, which is beneficial for the long-term preservation of the reagent. Further, the preservative is selected from one or more of sodium azide and ProClin 300.

[0047] In some specific examples, the concentration of the preservative was 0.0015 w / v% to 0.006 w / v%.

[0048] In some optional specific examples, the concentration of the preservative is 0.0015 w / v%, 0.0025 w / v%, 0.0035 w / v%, 0.0045 w / v%, or 0.006 w / v.

[0049] Furthermore, the concentration of the preservative is 0.0015 w / v% to 0.004 w / v%.

[0050] In some embodiments, the diluent includes a basal buffer, a coagulant, a surfactant, an alkali metal chloride, and an inert protein; wherein the basal buffer is selected from one of the following buffers: HEPES buffer, MOPS buffer, and PBS buffer; the coagulant is selected from one or more of polyethylene glycol 1000, polyethylene glycol 2000, polyethylene glycol 6000, and polyethylene glycol 8000; and the surfactant is selected from one or more of polyoxyethylene fatty alcohol ether-1, polyoxyethylene fatty alcohol ether-4, and polyoxyethylene fatty alcohol ether-5. The alkali metal chloride is selected from one or more of sodium chloride and calcium chloride, and the inert protein is selected from one or more of human serum albumin, bovine serum albumin, and horse serum albumin; the concentration of the basal buffer is 25 mmol / L to 50 mmol / L, the concentration of the coagulant is 0.02 w / v% to 0.1 w / v%, the concentration of the surfactant is 0.02 w / v% to 0.5 w / v%, the concentration of the alkali metal chloride is 0.5 w / v% to 1 w / v%, and the concentration of the inert protein is 0.1 w / v% to 0.5 w / v.

[0051] Furthermore, the diluents include a basal buffer, a coagulant, a surfactant, an alkali metal chloride, and an inert protein; the basal buffer is MOPS buffer, the coagulant is polyethylene glycol 2000, the surfactant is polyoxyethylene fatty alcohol ether-1, the alkali metal chloride is sodium chloride, and the inert protein is bovine serum albumin; the concentration of the basal buffer is 35 mmol / L to 50 mmol / L, the concentration of the coagulant is 0.02 w / v% to 0.05 w / v, the concentration of the surfactant is 0.03 w / v% to 0.08 w / v, the concentration of the alkali metal chloride is 0.8 w / v% to 1 w / v, and the concentration of the inert protein is 0.1 w / v% to 0.25 w / v.

[0052] Furthermore, the diluents include a basal buffer, a coagulant, a surfactant, an alkali metal chloride, and an inert protein; the basal buffer is MOPS buffer, the coagulant is polyethylene glycol 2000, the surfactant is polyoxyethylene fatty alcohol ether-1, the alkali metal chloride is sodium chloride, and the inert protein is bovine serum albumin; the concentration of the basal buffer is 50 mmol / L, the concentration of the coagulant is 0.02 w / v, the concentration of the surfactant is 0.05 w / v, the concentration of the alkali metal chloride is 1 w / v, and the concentration of the inert protein is 0.1 w / v.

[0053] In some embodiments, the diluent consists of a base buffer, a coagulant, a surfactant, an alkali metal chloride, an inert protein, and a preservative.

[0054] The diluent for the above-mentioned peroxidase conjugates, by adjusting the combined dosage of coagulant, surfactant and alkali metal chloride, achieves synergy among them through careful selection of dosage, so that the peroxidase conjugate diluent can effectively reduce background signal and improve signal-to-noise ratio while maintaining enzyme activity and protein stability.

[0055] In addition, this application provides a chemiluminescent immunoassay method, in which the peroxidase conjugate is diluted with the diluent of the peroxidase conjugate according to any of the above embodiments and then subjected to chemiluminescent immunoassay.

[0056] The peroxidase conjugate diluent of this application, when used in chemiluminescent immunoassay, has the following advantages: it can effectively reduce the background signal value, thereby improving the signal-to-noise ratio of the target analyte and enhancing the sensitivity and accuracy of chemiluminescent immunoassay. Specific Implementation

[0058] The present application will be further described below with reference to specific embodiments and comparative examples, but should not be construed as limiting the scope of protection of the present application.

[0059] Example 1

[0060] 1. Preparation of diluent

[0061] Prepare a 50 mmol MOPS buffer by mixing 0.2 g polyethylene glycol 6000, 0.5 g polyoxyethylene fatty alcohol ether-1, 10 g calcium chloride, 1 g bovine serum albumin, and 15 mg ProClin300 with the MOPS buffer, and then bringing the volume to 1 L with pure water and adjusting the pH to 7.0.

[0062] 2. Signal-to-noise ratio detection

[0063] (1) Instrument: Biochip analyzer manufactured by Jiangsu Sanlian Biotechnology Co., Ltd.

[0064] (2) The peroxidase conjugate was diluted using the diluent prepared in step 1 and the original diluent P1 (the original diluent P1 is the P1 type enzyme-labeled secondary antibody diluent produced by Jiangsu Sanlian Biotechnology Co., Ltd., P12206001). The quality control sample (positive signal) and the blocking sample (negative signal) were tested. Each sample was measured three times and the average value was taken. The test results are shown in Table 1.

[0065] Table 1. Detection results of background sample signals

[0066]

[0067] (3) The peroxidase conjugate was diluted using the diluent prepared in step (1) and the original diluent P1, respectively, and the following 12 samples were tested: pepsinogen I (PG I), pepsinogen II (PG II), carbohydrate antigen 199 (CA199), alpha-fetoprotein (AFP), neuron-specific enolase (NSE), human chorionic gonadotropin (HCG), cytokeratin fragment 19 (Cyfra21-1), carcinoembryonic antigen (CEA); carbohydrate antigen 724 (CA724), carbohydrate antigen 125 (CA125); gastrin precursor releasing peptide (GRP) and carbohydrate antigen 153 (CA153). The signal detection results of each sample are shown in Table 2 (average of 3 repeated experiments). The signal-to-noise ratio (i.e., the ratio of the positive signal value of each sample to the background positive signal value) was calculated, and the results are as follows: Figure 1 As shown.

[0068] Table 2. Signal detection results for different samples

[0069]

[0070] The above results show that, compared with the original diluent P1, the diluent of this application can significantly reduce the background signal value. Figure 1 As shown, the diluent for the peroxidase conjugate of this application can greatly improve the signal-to-noise ratio of each detection index compared with the original diluent P1.

[0071] Example 2

[0072] 1. Preparation of diluent

[0073] Prepare a 50 mmol PBS buffer by mixing 0.2 g polyethylene glycol 1000, 0.5 g polyoxyethylene fatty alcohol ether-5, 10 g sodium chloride, 1 g bovine serum albumin, and 15 mg ProClin300 with the PBS buffer to make a total volume of 1 L and adjusting the pH to 7.0.

[0074] 2. Stability Test

[0075] (1) Instrument: Biochip analyzer manufactured by Jiangsu Sanlian Biotechnology Co., Ltd.

[0076] (2) The diluent prepared in step 1 was placed at 37°C for 7 days for accelerated digestion. The peroxidase conjugate was diluted with the diluent before and after the acceleration, and the signal values ​​of the following 12 samples were detected: pepsinogen I (PG I), pepsinogen II (PG II), carbohydrate antigen 199 (CA199), alpha-fetoprotein (AFP), neuron-specific enolase (NSE), human chorionic gonadotropin (HCG), cytokeratin fragment 19 (Cyfra21-1), carcinoembryonic antigen (CEA), carbohydrate antigen 724 (CA724), carbohydrate antigen 125 (CA125), gastrin precursor releasing peptide (GRP), and carbohydrate antigen 153 (CA153). Each sample was measured twice, the average value was taken, and the deviation was calculated according to the following method:

[0077] Deviation = (Signal value after acceleration / Signal value before acceleration - 1) * 100%.

[0078] The test results are shown in Table 3 below.

[0079] Table 3. Results of diluent testing before and after acceleration in Example 2.

[0080]

[0081] The above results show that, after acceleration at 37°C for 7 days, the signal value deviation of the diluent for the peroxidase conjugate of this application is less than 10% compared with that of the diluent before acceleration; the original diluent P1, after acceleration at 37°C for 7 days, shows a significantly higher signal value deviation than the diluent of this application. This indicates that the diluent of this application can maintain enzyme activity and protein stability, ensuring the accuracy and reliability of the detection results.

[0082] Example 3

[0083] 1. Preparation of diluent

[0084] Prepare a 50 mmol MOPS buffer by mixing 0.2 g polyethylene glycol 1000, 0.5 g polyoxyethylene fatty alcohol ether-4, 10 g sodium chloride, 1 g bovine serum albumin, and 15 mg ProClin300 with the MOPS buffer to make a total volume of 1 L and adjusting the pH to 7.0.

[0085] 2. Daytime precision testing

[0086] (1) Instrument: Biochip analyzer manufactured by Jiangsu Sanlian Biotechnology Co., Ltd.

[0087] (2) Dilute the peroxidase conjugate with the diluent prepared in step 1 and test the following 12 samples: pepsinogen I (PG I), pepsinogen II (PG II), carbohydrate antigen 199 (CA199), alpha-fetoprotein (AFP), total prostate-specific antigen (T-PSA), free prostate-specific antigen (fPSA), neuron-specific enolase (NSE), cytokeratin fragment 19 (Cyfra21-1), carbohydrate antigen 125 (CA125), carcinoembryonic antigen (CEA), carbohydrate antigen 724 (CA724), and progastrin-releasing peptide (Pro GRP). Each sample was tested on the morning, afternoon, and morning of the same day, and the coefficient of variation (CV) was calculated based on the results of the three tests. The results are shown in Table 4.

[0088] Table 4. Calculation results of the coefficient of variation of the diluent in Example 3.

[0089]

[0090]

[0091] The above results indicate that the coefficient of variation of the peroxidase conjugate diluent of this application in daytime repeat tests does not exceed 15%, which is not significantly different from the coefficient of variation of the original diluent.

[0092] Example 4

[0093] 1. Preparation of diluent

[0094] Prepare a 40 mmol PBS buffer by mixing 0.4 g polyethylene glycol 2000, 8 g calcium chloride, 2 g horse serum albumin, and 15 mg ProClin 300 with the PBS buffer, and then adding 0.5 g each of polyoxyethylene fatty alcohol ether-1, polyoxyethylene fatty alcohol ether-2, polyoxyethylene fatty alcohol ether-3, polyoxyethylene fatty alcohol ether-4, and polyoxyethylene fatty alcohol ether-5 as surfactants, making the total volume 1 L, and adjusting the pH to 7.0 to obtain diluents 1–5.

[0095] 2. Performance testing of different surfactants

[0096] (1) Instrument: Biochip analyzer manufactured by Jiangsu Sanlian Biotechnology Co., Ltd.

[0097] (2) After diluting the peroxidase conjugate with the five diluents prepared in step 1, the following 12 samples were tested: pepsinogen I (PG I), pepsinogen II (PG II), carbohydrate antigen 199 (CA199), alpha-fetoprotein (AFP), neuron-specific enolase (NSE), human chorionic gonadotropin (HCG), cytokeratin fragment 19 (Cyfra21-1), carcinoembryonic antigen (CEA), carbohydrate antigen 724 (CA724), carbohydrate antigen 125 (CA125), gastrin precursor releasing peptide (GRP), and carbohydrate antigen 153 (CA153). Each sample was tested three times, and the average value was calculated. The results are shown in Table 5. Figure 2 The image shows the signal-to-noise ratio analysis results for the five diluents in this embodiment.

[0098] Table 5. Signal detection results of diluents with different surfactants added.

[0099]

[0100] As can be seen from this embodiment, in the diluents containing polyoxyethylene fatty alcohol ether-1, polyoxyethylene fatty alcohol ether-2, polyoxyethylene fatty alcohol ether-3, polyoxyethylene fatty alcohol ether-4, and polyoxyethylene fatty alcohol ether-5 as surfactants, diluent 1 with polyoxyethylene fatty alcohol ether-1 showed the best effect in improving the signal-to-noise ratio, followed by polyoxyethylene fatty alcohol ether-4 and polyoxyethylene fatty alcohol ether-5; polyoxyethylene fatty alcohol ether-3 was next, while polyoxyethylene fatty alcohol ether-2 was relatively poor. The above results indicate that the diluents selected in this application, with the addition of polyoxyethylene fatty alcohol ether-1, polyoxyethylene fatty alcohol ether-4, and polyoxyethylene fatty alcohol ether-5, can effectively improve the signal-to-noise ratio of chemiluminescence detection.

[0101] Example 5

[0102] 1. Preparation of diluent

[0103] Prepare a 45 mmol MOPS buffer solution by mixing 0.2 g polyethylene glycol 8000, 8 g sodium chloride, 2 g horse serum albumin, and 15 mg ProClin300 with the MOPS buffer solution. Add different concentrations of polyoxyethylene fatty alcohol ether-1 as a surfactant (the highest concentration is 1.5 w / v%) and dilute in half sequentially to make a total volume of 1 L. Adjust the pH to 7.0 to obtain 12 different concentrations of polyoxyethylene fatty alcohol ether-1 diluents.

[0104] 2. Concentration screening of surfactants

[0105] (1) Instrument: Biochip analyzer manufactured by Jiangsu Sanlian Biotechnology Co., Ltd.

[0106] (2) After diluting the peroxidase conjugate with the 12 diluents prepared in step 1, the positive signal of the background sample was tested. Each sample was measured three times. The test results are as follows: Figure 3 As shown.

[0107] Please see Figure 3 As the concentration of the surfactant increases, the background signal value decreases. When the concentration reaches 0.02 w / v% to 0.5 w / v%, the downward trend of the background signal slows down, and the effect is not obvious. This indicates that the surfactant in the diluent of this application can effectively reduce the background signal value at 0.02 w / v% to 0.5 w / v%, without affecting the signal value of the target analyte.

[0108] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0109] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A diluent for a peroxidase conjugate, characterized in that, The formulation includes a basal buffer, a coagulant, a surfactant, an alkali metal chloride, and an inert protein; the surfactant is selected from one or more of polyoxyethylene fatty alcohol ether-1, polyoxyethylene fatty alcohol ether-4, and polyoxyethylene fatty alcohol ether-5; the concentration of the coagulant is 0.02 w / v% to 0.1 w / v%; the concentration of the surfactant is 0.02 w / v% to 0.5 w / v%; the concentration of the alkali metal chloride is 0.5 w / v% to 1 w / v%; and the concentration of the inert protein is 0.1 w / v% to 0.5 w / v.

2. The diluent for the peroxidase conjugate according to claim 1, characterized in that, The concentration of the surfactant is 0.03 w / v% to 0.08 w / v%; and / or, The concentration of the solute in the basal buffer solution is 25 mmol / L to 50 mmol / L.

3. The diluent for the peroxidase conjugate according to claim 1, characterized in that, The pH value of the diluent is 6.0 to 7.

0.

4. The diluent for the peroxidase conjugate according to any one of claims 1 to 3, characterized in that, The base buffer is selected from one or more of the following buffers: HEPES buffer, MOPS buffer, and PBS buffer.

5. The diluent for the peroxidase conjugate according to any one of claims 1 to 3, characterized in that, The coagulant is selected from one or more of polyethylene glycol 1000, polyethylene glycol 2000, polyethylene glycol 6000 and polyethylene glycol 8000.

6. The diluent for the peroxidase conjugate according to any one of claims 1 to 3, characterized in that, The surfactant is selected from one or more of polyoxyethylene fatty alcohol ether-1, polyoxyethylene fatty alcohol ether-4, and polyoxyethylene fatty alcohol ether-5.

7. The diluent for the peroxidase conjugate according to any one of claims 1 to 3, characterized in that, The alkali metal chloride is selected from one or more of sodium chloride and calcium chloride.

8. The diluent for the peroxidase conjugate according to any one of claims 1 to 3, characterized in that, The inert protein is selected from one or more of human serum albumin, bovine serum albumin, and horse serum albumin.

9. The diluent for the peroxidase conjugate according to any one of claims 1 to 3, characterized in that, The diluent also includes a preservative selected from one or more of sodium azide and ProClin 300.

10. A chemiluminescent immunoassay method, characterized in that, The peroxidase conjugate was diluted with the diluent of any one of claims 1 to 9 and then subjected to chemiluminescent immunoassay.

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