Light-activated chemiluminescence immunodetection method for rapidly quantifying C-reactive protein

Abstract

The invention discloses a light-activated chemiluminescence immunodetection method for rapidly quantifying C-reactive protein (CRP), and belongs to the technical field of marker detection. Comprising a luminous microsphere-CRP coated antibody complex, a biotin-CRP labeled antibody complex and a streptavidin coated photosensitive microsphere. The light-sensitive microspheres and the light-emitting microspheres are ingeniously combined, and key factors such as coupling efficiency of antibodies on the surfaces of the light-emitting microspheres, reagent dosage, incubation temperature and time and the like are elaborately optimized, so that an optimized light-activated chemiluminescence detection system is constructed. The system not only simplifies the experimental process, but also ensures the reliability and consistency of the detection result by enhancing the stability and specificity of signal transduction. By screening and optimizing conditions of the system, the invention successfully develops a novel CRP (C-reactive protein) light-activated chemiluminescence immunoassay kit, and the detection limit is 6.20 ng.mL <-1 >.

Description

Technical Field

[0001] The present invention relates to the technical field of marker detection, and in particular to a light-induced chemiluminescence immunoassay method for rapid quantitative C-reactive protein. Background Art

[0002] Serum C-reactive protein (CRP) is an indispensable inflammatory marker in clinical practice. Its level changes are of great significance for assessing the type and severity of inflammatory infection and predicting the risk of cardiovascular disease. Although there are many CRP detection kits on the market, these products have made significant progress in sensitivity and specificity, but most of them still rely on heterogeneous detection systems. Their complex operation procedures and frequent separation and washing steps are not only time-consuming and labor-intensive, but may also lead to deviations in test results and accumulation of errors. Therefore, it is crucial to provide an efficient and convenient CRP detection method. Summary of the invention

[0003] The purpose of the present invention is to provide a photochemiluminescence immunoassay method for rapid quantitative C-reactive protein to solve the problems existing in the above-mentioned prior art. The present invention innovatively focuses on the development of homogeneous immunoassay technology, especially by designing specific microspheres and introducing a novel mechanism based on singlet oxygen signal transduction, aiming to create an efficient and convenient CRP detection platform. This platform will achieve rapid, accurate and quantitative detection of CRP protein without cumbersome separation and washing steps, thereby significantly improving detection efficiency and accuracy.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] One of the technical solutions of the present invention is a C-reactive protein detection system, which includes a luminescent microsphere-CRP coated antibody complex, a biotin-CRP labeled antibody complex and a streptavidin-coated photosensitive microsphere.

[0006] The second technical solution of the present invention is a method for detecting C-reactive protein for non-disease diagnosis or treatment purposes, comprising the following steps:

[0007] (1) mixing the sample to be tested with the luminescent microsphere-CRP coated antibody complex and the biotin-CRP labeled antibody complex for the first incubation;

[0008] (2) After the incubation is completed, streptavidin-coated photosensitive microspheres are added for a second incubation;

[0009] (3) Determine the content of C-reactive protein in the sample based on the CL value after the second incubation.

[0010] Based on the above technical solution, the present invention has the following technical effects:

[0011] The present invention uses a clever combination of photosensitive microspheres (GG) and luminescent microspheres (FG), and constructs an optimized photochemiluminescence detection system by carefully optimizing key factors such as the coupling efficiency of the antibody on the surface of the luminescent microspheres, the amount of reagents, and the incubation temperature and time. This system not only simplifies the experimental process, but also ensures the reliability and consistency of the test results by enhancing the stability and specificity of signal transduction. Through systematic condition screening and optimization, the present invention successfully developed a new CRP photochemiluminescence immunoassay kit with a detection limit of 6.20ng·mL -1 . Provide strong technical support for the rapid diagnosis, disease monitoring and prognosis evaluation of clinical inflammatory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0013] Figure 1 To study the optimal mass ratio for coupling luminescent microspheres with CRP-coated antibodies.

[0014] Figure 2 To study the optimal volume ratio of luminescent microspheres-CRP coated antibody complex and diluent.

[0015] Figure 3 For laser power research.

[0016] Figure 4 For irradiation time study.

[0017] Figure 5 This study is to study the volume ratio of luminescent microsphere-CRP coated antibody complex and biotin-CRP labeled antibody complex in CRP-Ag detection.

[0018] Figure 6 This study aims to investigate the volume ratio of luminescent microspheres-CRP-coated antibody complexes to streptavidin-coated photosensitive microspheres in CRP-Ag detection.

[0019] Figure 7 This study is to study the volume ratio of luminescent microspheres-CRP coated antibody complex and standard in CRP-Ag detection.

[0020] Figure 8 Optimize for incubation temperature.

[0021] Fig. 9 Optimize for incubation time.

[0022] Fig.10 This is the effect of the number of consecutive tests on the CL value.

[0023] Fig.11 Specificity for CRP antibodies.

[0024] Fig.12 This is the HOOK effect in CRP detection.

[0025] Fig.13 Response spectrum of CRP-Ag; Inset: linear relationship between CRP-Ag concentration and CL value.

[0026] Fig.14 The anti-interference ability of CRP antibodies.

[0027] Fig.15 This is the principle diagram of photochemiluminescence detection. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0034] The embodiment of the present invention provides a C-reactive protein detection system, including a luminescent microsphere-CRP coated antibody complex, a biotin-CRP labeled antibody complex and a streptavidin-coated photosensitive microsphere.

[0035] In some specific embodiments, the preparation method of the luminescent microsphere-CRP coated antibody complex comprises: mixing the activated luminescent microspheres with the C-reactive protein coated antibodies at a mass ratio of 2:5 to form the luminescent microsphere-CRP coated antibody complex.

[0036] In some specific embodiments, the preparation method of the biotin-CRP labeled antibody complex comprises: concentrating the CRP labeled antibody and removing impurities, then mixing the CRP labeled antibody solution with the biotin solution at a volume concentration ratio of 1:50, and incubating to obtain the biotin-CRP labeled antibody;

[0037] The concentration of the biotin solution is 10 mmol·L -1 ;

[0038] The concentration of the CRP coated antibody solution is 500ug·mL -1 ;

[0039] The concentration of the CRP labeled antibody solution is 2.5 mg·mL -1 ;

[0040] The concentration and impurity removal treatment method is dialysis or ultrafiltration tube treatment.

[0041] In some specific embodiments, the volume ratio of the luminescent microsphere-CRP coated antibody complex, the biotin-CRP labeled antibody complex and the streptavidin coated photosensitive microsphere is 1:1:2.

[0042] The present invention also provides a method for detecting C-reactive protein for non-disease diagnosis or treatment purposes, comprising the following steps:

[0043] (1) mixing the sample to be tested with the luminescent microsphere-CRP coated antibody complex and the biotin-CRP labeled antibody complex for the first incubation;

[0044] (2) After the incubation is completed, streptavidin-coated photosensitive microspheres are added for a second incubation;

[0045] (3) Determine the content of C-reactive protein in the sample based on the CL value after the second incubation.

[0046] In some specific embodiments, the volume ratio of the sample to be tested to the luminescent microsphere-CRP coated antibody complex is 1:4.

[0047] In some specific embodiments, the conditions for measuring the CL value are: laser power is 110 mW, and irradiation time is 1200 ms.

[0048] In some specific embodiments, the conditions of the first incubation and the second incubation are: incubation at 37° C. for 5 min.

[0049] Example 1

[0050] 1. Preparation of buffer solution

[0051] (1) Activation buffer: 0.1 mol·L -1 MES buffer, pH adjusted to 6.0.

[0052] (2) Buffer for coupling luminescent microspheres and antibodies: 0.1 mol·L -1 Borate buffer, pH adjusted to 8.0.

[0053] (3) Storage buffer: 0.1 mol·L -1 PBS buffer, containing 0.05% Proclin-300, adjusted to pH 7.4.

[0054] (4) Biotin and antibody connection buffer: 0.1 mol·L -1 Na2CO3 / NaHCO3 buffer, containing 0.1% sodium azide, adjusted to pH 9.5.

[0055] (5) Washing buffer: It is composed of 0.1 M sodium carbonate and 0.1 M sodium bicarbonate, with a pH range of 9.2 to 10.4.

[0056] 2 Direct coupling of luminescent microspheres and CRP-coated antibodies to prepare luminescent microsphere-CRP-coated antibody complex (FG-CRP)

[0057] 1) Washing: Take 100 μL of luminescent microspheres (10 mg mL -1 ) to contain 1 mL of activation buffer in a 2 mL centrifuge tube, mix by ultrasonication for 7-8 seconds, centrifuge at 14000 rpm, 15°C for 15 min, and remove the supernatant.

[0058] 2) Activation: Add 1 mL of activation buffer, sonicate for 7-8 seconds, remove and mix thoroughly, repeat 7-8 times; first add 7 μL (10 mg mL) of activating agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) -1 ), oscillate and mix, then add 66 μL (10 mg mL -1 ), shake and mix, seal with sealing film, turntable to protect from light, 40r / min, 25℃, and activate for 15min.

[0059] 3) Cleaning: After activation, centrifuge at 14000rpm, 15℃ for 15min, and remove the supernatant. Add 1mL of activation buffer, sonicate for 7-8s, remove and mix by inversion, and repeat this process 7-8 times. Centrifuge at 1400rpm, 15℃ for 15min, and remove the supernatant. Then centrifuge at 14000rpm, 15℃ for 15min, and remove the supernatant. Add 1mL of deionized water, sonicate for 7-8s, remove and mix by inversion, and repeat this process 7-8 times. Centrifuge at 14000rpm, 15℃ for 15min, and remove the supernatant.

[0060] 4) Coupling: Add 0.2 mL of coupling buffer to the cleaned luminescent microspheres, mix by ultrasonication for 5 seconds, then add 0.8 mL of coupling buffer, ultrasonicate for 7-8 seconds, remove and mix by inversion, and repeat this process 7-8 times. Carefully add 4.5 μL of 0.5 mg·mL -1 C-reactive protein (CRP) was coated with antibody (Huakui Jinpei Biotechnology Co., Ltd., model number M30311) and mixed by oscillation. After mixing by oscillation, the tube was sealed with sealing film and reacted on a turntable in the dark at 40 r / min and 25°C for 2 h.

[0061] 5) Blocking and washing: After the coupling is completed, add 0.1mL of fast blocking solution (bovine serum albumin BSA), shake and mix, then seal the film and turntable to avoid light reaction, 40r / min 25℃ blocking for 2h or overnight blocking for 12-16h. After the blocking is completed, centrifuge to remove the supernatant, first add 0.2mL of washing buffer, ultrasonically mix for 5s, then add 0.8mL of washing buffer, ultrasonically mix for 7-8s, take out and mix by inversion, repeat 7-8 times, centrifuge to remove the supernatant, and then wash repeatedly 3 times.

[0062] 6) Storage: Finally, add 0.2 mL of storage buffer, mix by ultrasonic for 5 seconds, then add 0.8 mL of storage buffer, ultrasonic for 7-8 seconds, remove and mix by inversion, and repeat 7-8 times; the final concentration is 1 mg·mL -1 , store at 2-8℃ for later use.

[0063] 3. Biotin and CRP labeled antibody (Huakui Jinpei Biotechnology Co., Ltd., model M30312) were connected to prepare biotin-CRP labeled antibody complex (Bi-CRP)

[0064] (1) Pretreatment with CRP-labeled antibodies

[0065] Since the buffer used to protect the CRP labeled antibody usually contains sodium azide (NaN3) or primary amine, these substances will affect the detection and must be removed. At the same time, the antibody must reach a certain concentration, so the labeled antibody must first be concentrated and impurities removed. There are currently two treatment methods: dialysis and ultrafiltration tube. This experiment uses the ultrafiltration tube method. The specific pretreatment steps are as follows:

[0066] Ultrafiltration tube pretreatment: The ultrafiltration tube needs to be cleaned before use. First, rinse the filter with ultrapure water; then rinse with 0.1 mol·L -1 The samples were rinsed with sodium hydroxide and then washed with ultrapure water and ligation buffer (0.1 mol·L -1 NaHCO3, pH 8.3) for subsequent cleaning and drying;

[0067] Concentration of CRP labeled antibody: Place the antibody in an ultrafiltration centrifuge tube with a molecular weight cutoff of 10KDa, and repeat ultrafiltration and centrifugation 3 times in the connection buffer;

[0068] Collection of CRP labeled antibodies: Replace with a new collection tube, turn the ultrafiltration tube upside down, centrifuge at 8000g for 3 minutes, and collect the resulting filtrate (i.e., the required antibody).

[0069] (2) Conjugation of biotin and CRP-labeled antibody

[0070] Take out the biotin reagent (Beijing Boxingyuan Biotechnology Co., Ltd., catalog number BXY20240101) from the refrigerator and equilibrate it to room temperature before use. Prepare 10 mmol·L -1 According to the instructions of the biotin reagent, CRP labeled antibody solution (2.5 mg mL -1 ) was mixed with biotin solution at a volume concentration ratio of 1:50 and incubated at room temperature for 1 hour. Excess biotin was removed using a JET Ultra-15 ultrafiltration centrifuge tube. After centrifugation at 5000g for 30 minutes, the supernatant was removed, and a certain amount of washing buffer was added. The precipitate was resuspended and this step was repeated 3 times. Finally, the precipitate was resuspended with the storage buffer to obtain the biotin-CRP labeled antibody complex. The concentration of the biotin-CRP labeled antibody complex was determined using the BCA protein quantitative analysis reagent and adjusted to 0.2 mg mL -1 .

[0071] 4 Study on CRP antigen (CRP-Ag) detection based on photochemiluminescence immunoassay

[0072] The photochemiluminescent immunoassay follows a two-step reaction. The experiments were performed in duplicate using calibrators and samples. For the general assay procedure, 10 μL of the test serum sample was added to a 96-well plate. Then 20 μL of the luminescent microsphere-CRP coated antibody complex and 20 μL of the biotin-CRP labeled antibody complex were added. For the standard curve, the procedure was the same, except that the serum samples were changed to serial dilutions of different concentrations of the standard. The plate was incubated at 37°C for 6 minutes and gently stirred on a rocking platform. Finally, 40 μL of streptavidin-coated photosensitive microspheres (SA-GG, purchased from Degree Biotech) were added to the reaction mixture in all wells and incubated for another 6 minutes at 37°C in dark conditions. The CL intensity was then measured on a LICA reader.

[0073] 4.1 Optimization of the amount of conjugation between luminescent microspheres and CPR-coated antibodies

[0074] In 1mg·mL -1 The surface of the luminescent microspheres (20 μL) was coupled with 2, 4, 6, 8, 10 and 12 μL of CRP (0.5 mg mL -1 ) were coated with antibodies at mass ratios of 2:1; 1:1; 2:3; 1:2; 2:5; 1:3. 10 μL of 200 ng·mL -1 The CRP-Ag standard was added to a 96-well plate. Then 20 μL of the luminescent microsphere-CRP coated antibody complex obtained by coupling at different mass ratios and 20 μL of the biotin-CRP labeled antibody complex were added. The plate was incubated at 37°C for 6 minutes and gently stirred on a rocking platform. Finally, 40 μL of streptavidin-coated photosensitive microspheres were added to the reaction mixture in all wells and incubated for another 6 minutes at 37°C in the dark. The CL intensity was then measured on a LICA reader. The results are shown in Figure 2. Figure 1 As can be seen from the figure, as the volume of the coupled CRP-coated antibody increases, the CL value generally shows a trend of increasing first and then decreasing. When the coupled antibody volume is 10 μL, the CL value is the largest, indicating that the coupling efficiency of the microspheres and antibodies is the highest at this time, so 10 μL is selected as the optimal coupling volume. That is, the mass ratio is 2:5. Subsequent studies have used the luminescent microsphere-CRP-coated antibody complex coupled with this mass ratio.

[0075] 4.2 Optimization of the dilution volume ratio of luminescent microsphere-CRP coated antibody complex (coupling mass ratio of 2:5) and diluent

[0076] The CL values ​​of the luminescent microsphere-CRP coated antibody complex and the diluent at different dilution volume ratios (Dope, 1:9, 1:19, 1:29, 1:39, 1:49) were compared.

[0077] 10 μL 200 ng mL -1 The CRP-Ag standard was added to a 96-well plate. Then, 20 μL of the luminescent microsphere-CRP coated antibody complex and 20 μL of the biotin-CRP labeled antibody complex diluted in different volume ratios were added. The plate was incubated at 37°C for 6 minutes and gently stirred on a rocking platform. Finally, 40 μL of streptavidin-coated photosensitive microspheres were added to the reaction mixture in all wells and incubated for another 6 minutes at 37°C in the dark. The CL intensity was then measured on a LICA reader. The results are shown in Figure 2. Figure 2 It can be seen that with the increase of the dilution volume ratio of the luminescent microsphere-CRP coated antibody complex, the CL value first increases and then decreases. When the dilution volume ratio is 1:39, the luminescent signal value is higher and the blank value is lower. Therefore, the optimal dilution volume ratio is 1:39, that is, 40 times dilution, and the final concentration is 0.025 mg·mL -1 All subsequent studies were conducted at this concentration.

[0078] 4.3 Optimization of laser power and irradiation time

[0079] The CL values ​​measured at different laser powers (80mW, 90mw, 100mW, 110mW, 120mW) and irradiation times (600ms, 800ms, 1000ms, 1200ms, 1400ms) were compared.

[0080] 10 μL 200 ng mL -1 The CRP-Ag standard was added to a 96-well plate. Then 20 μL of luminescent microsphere-CRP coated antibody complex (0.025 mg mL -1 ) and 20 μL of biotin-CRP labeled antibody complex. The plate was incubated at 37°C for 6 minutes with gentle agitation on a rocking platform. Finally, 40 μL of streptavidin-coated photosensitive microspheres were added to the reaction mixture in all wells and incubated for another 6 minutes at 37°C in the dark. Only the laser power or irradiation time was changed to measure the CL intensity on the LICA reader. The results are shown in Figure 3 , 4 As shown in the figure, it can be seen that when the laser power is 110mW and the irradiation time is 1200ms, the CL value reaches the highest, and the chemiluminescence signal does not change much when the power and time continue to increase, so it is believed that the optimal laser power and the optimal irradiation time are 110mW and 1200ms respectively. Subsequent studies were carried out under this detection condition.

[0081] 4.4 Study on the optimal volume ratio of luminescent microspheres-CRP-coated antibody complexes and streptavidin-coated photosensitive microspheres

[0082] The CL values ​​were detected and compared at the volume ratios of luminescent microspheres-CRP-coated antibody complexes and streptavidin-coated photosensitive microspheres (None, 2:1, 1:1, 2:3, 1:2, 2:5).

[0083] 10 μL 200 ng mL -1 The CRP-Ag standard was added to a 96-well plate. Then 20 μL of luminescent microsphere-CRP coated antibody complex and 20 μL of biotin-CRP labeled antibody complex were added. The plate was incubated at 37°C for 6 minutes and gently stirred on a rocking platform. Finally, different volumes (0, 10, 20, 30, 40, 50 μL) of streptavidin-coated photosensitive microspheres were added to the reaction mixture in all wells and incubated for another 6 minutes at 37°C in the dark. The CL intensity was then measured on a LICA reader to examine the optimal volume ratio of the luminescent microsphere-CRP coated antibody complex and the streptavidin-coated photosensitive microspheres. The results are shown in Figure 2. Figure 5 As shown in the figure, as the volume of the photosensitive microspheres coated with streptavidin increases, the CL value increases first and then decreases. When the volume ratio of the luminescent microsphere-CRP-coated antibody complex to the photosensitive microspheres coated with streptavidin is 1:2, the CL value is the largest, and this ratio is considered to be the optimal volume ratio, that is, the volume of the photosensitive microspheres coated with streptavidin is 40 μL. All subsequent studies were conducted under this volume ratio.

[0084] 4.5 Study on the Optimal Volume Ratio of Luminescent Microsphere-CRP Coated Antibody Complex and Biotin-CRP Labeled Antibody Complex

[0085] When the volume ratio of the luminescent microsphere-CRP coated antibody complex to the biotin-CRP labeled antibody complex was (None, 2:1, 1:1, 2:3, 1:2, 2:5), the CL values ​​measured at different ratios were compared.

[0086] 10 μL 200 ng mL -1The CRP-Ag standard was added to a 96-well plate. Then 20 μL of luminescent microsphere-CRP coated antibody complex and different volumes (0, 10, 20, 30, 40, 50 μL) of biotin-CRP labeled antibody complex were added. The plate was incubated at 37°C for 6 minutes and gently stirred on a rocking platform. Finally, 40 μL of streptavidin-coated photosensitive microspheres were added to the reaction mixture in all wells and incubated for another 6 minutes at 37°C in the dark. The CL intensity was then measured on the LICA reader to examine the optimal volume ratio of the luminescent microsphere-CRP coated antibody complex to the biotin-CRP labeled antibody complex during the detection process. The results are shown in Figure 2. Figure 6 As shown in the figure, it can be seen that with the increase of the volume of the biotin-CRP labeled antibody complex, the CL value first increases and then decreases, and when the volume ratio of the luminescent microsphere-CRP coated antibody complex to the biotin-CRP labeled antibody complex is 1:1, the CL value is the largest, that is, the volume ratio of the luminescent microsphere-CRP coated antibody complex to the biotin-CRP labeled antibody complex is 1:1. All subsequent studies were carried out under this volume ratio.

[0087] 4.6 Study on the Optimal Volume Ratio of Luminescent Microsphere-CRP Coated Antibody Complex and CRP-Ag Standard

[0088] When 20 μL of luminescent microsphere-CRP coated antibody complex was mixed with 200 ng·mL -1 The volume ratios of the CRP-Ag standard samples were (5:1, 4:1, 2:1, 1:1, 4:5), and the signal values ​​measured at different ratios were compared.

[0089] Similarly, 4, 5, 10, 20, and 25 μL of 200 ng mL -1 CRP-Ag standard. Then add 20μL of luminescent microsphere-CRP coated antibody complex and 20μL of biotin-CRP labeled antibody complex. Incubate the plate at 37°C for 6 minutes and gently stir on a rocking platform. Finally, add 40μL of streptavidin-coated photosensitive microspheres to the reaction mixture in all wells and incubate for another 6 minutes at 37°C in the dark. The CL intensity is then measured on a LICA reader to investigate the optimal volume ratio of the luminescent microsphere-CRP coated antibody complex to the CRP-Ag standard. The results are as follows: Figure 7 As shown in the figure, it can be seen that as the volume of CRP-Ag standard increases, the CL value increases first and then decreases, and when the volume ratio of luminescent microsphere-CRP coated antibody complex to CRP-Ag standard is 4:1, the CL value is the largest, so the optimal volume ratio of luminescent microsphere-CRP coated antibody complex to CRP-Ag standard is 4:1, that is, the volume of CRP-Ag standard is 5μL. All subsequent studies were carried out under this volume ratio.

[0090] 4.7 Optimization of reaction temperature (15°C, 25°C, 37°C, 42°C)

[0091] The reaction temperatures of the first and second stages of the CRP photochemiluminescence method were 15°C, 25°C, 37°C, and 42°C, respectively, to monitor and compare the light signal values.

[0092] 5 μL of 200 ng mL -1 The CRP-Ag standard was added to a 96-well plate, followed by the addition of 20 μL of luminescent microspheres-CRP coated antibody complex and 20 μL of biotin-CRP labeled antibody complex. The plate was incubated at 15°C, 25°C, 37°C, and 42°C for 6 minutes, respectively, and gently stirred on a rocking platform. Finally, 40 μL of streptavidin-coated photosensitive microspheres were added to the reaction mixture in all wells, and incubated for another 6 minutes in the dark under the same temperature conditions. The CL intensity was then measured on a LICA reader to examine the optimal incubation temperature. The results are as follows: Figure 8 As shown in the figure, it can be seen that with the increase of incubation temperature, the CL value shows an increasing trend. When the incubation temperature is 37°C, the CL value increases the most. Therefore, the optimal incubation temperature and time for CRP-Ag detection is 37°C. All subsequent studies were carried out at this incubation temperature.

[0093] 4.8 Optimization of two-stage reaction time (15s, 1min, 5min, 10min)

[0094] The first and second stage reaction times of the CRP photochemiluminescence method were 15s, 1min, 5min, and 10min, respectively, to monitor and compare the light signal values.

[0095] 5 μL of 200 ng mL -1 The CRP-Ag standard was added to a 96-well plate, followed by the addition of 20 μL of luminescent microspheres-CRP coated antibody complex and 20 μL of biotin-CRP labeled antibody complex. The plate was incubated at 37°C for different times (15s, 1min, 5min, 10min) and gently stirred on a rocking platform. Finally, 40 μL of streptavidin-coated photosensitive microspheres were added to the reaction mixture in all wells and incubated again at 37°C in the dark for the same time. The CL intensity was then measured on a LICA reader to investigate the optimal incubation time. The results are shown in Figure 2. Fig. 9 As shown in the figure, it can be seen that with the increase of incubation time, the CL value shows an increasing trend. When the incubation time is 5 minutes, the CL value of the higher CRP-Ag standard concentration is the largest. When the time is increased, the signal decreases. Therefore, the optimal incubation time in CRP-Ag detection is 5 minutes. All subsequent studies were carried out under this incubation time.

[0096] 4.9 Study the effect of consecutive detection times on CL value

[0097] 5 μL of 100 and 200 ng mL -1 The CRP-Ag standard was added to a 96-well plate, followed by the addition of 20 μL of luminescent microspheres-CRP coated antibody complex and 20 μL of biotin-CRP labeled antibody complex. The plate was incubated at 37°C for 5 minutes and gently stirred on a rocking platform. Finally, 40 μL of streptavidin-coated photosensitive microspheres were added to the reaction mixture in all wells and incubated for another 5 minutes at 37°C in the dark. The CL intensity was then measured on a LICA reader, and after the first detection, a total of 6 consecutive detections were performed at intervals of 15 seconds to obtain the CL value for each detection. The results are shown in the figure. Fig.10 As shown in the figure, it can be seen that when the detection interval is 15s, 6 consecutive detections are performed. As the number of detections increases, the CL value shows a decreasing trend, and the working curve of the number of detections is linear. 2 They are 0.9148 and 0.9442 respectively.

[0098] Example 2 Preliminary clinical application

[0099] 1. Sample pretreatment

[0100] 1.1 Collect fresh venous blood (2-5 mL) into a red cap blood collection tube (without anticoagulant) and let it stand at room temperature for 30-60 minutes or at 2-8°C overnight until the blood coagulates;

[0101] 1.2 Centrifugation: 3500 rpm, 5 min;

[0102] 1.3 Sampling: extract the upper serum for testing;

[0103] 1.4 Storage: Store at 2-8℃ for 24h, or below -15℃ for 90 days.

[0104] 1.5 Detection: Add 5 μL of test serum sample to the wells of a 96-well microtiter plate. Then add 20 μL of luminescent microsphere-CRP coated antibody complex and 20 μL of biotin-CRP labeled antibody complex. Incubate the plate at 37°C for 5 minutes and gently stir on a shaking platform. Finally, add 40 μL of streptavidin-coated photosensitive microspheres to the reaction mixture in all wells and incubate for another 5 minutes at 37°C in the dark. Then measure the CL intensity on a LICA reader.

[0105] 2 Specificity analysis

[0106] Prepare a concentration of 200 ng mL -1 CRP-Ag standard and 1000ng·mL -1Antigen standards (IL-6, cTnT, P24, tPAI·C, SAA, PCT) were prepared and stored under frozen conditions for future use. 5 μL of the above antigen standards were taken into a 96-well plate, and then 20 μL of luminescent microspheres-CRP coated antibody complex and 20 μL of biotin-CRP labeled antibody complex were added. The plate was incubated at 37°C for 5 minutes and gently stirred on a rocking platform. Finally, 40 μL of streptavidin-coated photosensitive microspheres were added to the reaction mixture in all wells and incubated for another 5 minutes at 37°C in the dark. The CL intensity was then measured on a LICA reader. The results are shown in the figure. Fig.11 As shown in the figure, the CL value changes the most after adding CRP-Ag, while other antigens hardly change. This phenomenon shows that the analysis method has good specificity for CRP-Ag. This strong specificity provides an important theoretical basis for the establishment of photochemiluminescence analysis method and the determination of clinical serum samples.

[0107] 3. Drawing of standard curve and HOOK effect

[0108] The CRP-Ag standard was diluted with PBS buffer to different concentrations for determination, with the concentrations being 0.021, 0.086, 0.21, 1.02, 2.15, 4.3, and 21 μg mL -1 , a total of 8 concentration values, the CL value was measured using the LICA reader according to the same experimental operation as before. Fig.12 As shown, this detection method has an antigen concentration of 0-21ug·mL -1 No HOOK effect was observed.

[0109] CRP-Ag standards were diluted to 0.021, 0.086, 0.21, 1.02, 2.15 and 4.3 μg mL -1 Different concentrations were measured. 5 μL of CRP-Ag standards of different concentrations were added to the wells of a 96-well microtiter plate. Then 20 μL of luminescent microspheres-CRP coated antibody complex and 20 μL of biotin-CRP labeled antibody complex were added. The plate was incubated at 37 °C for 5 minutes and gently stirred on a rocking platform. Finally, 40 μL of streptavidin-coated photosensitive microspheres were added to the reaction mixture in all wells and incubated for another 5 minutes at 37 °C in the dark. The CL intensity was then measured on a LICA reader. The results are shown in Figure 2. Fig.13 As shown, the linear equation is y = 2110.77x-14.53, R 2 =0.9946, linear range is 0.021~4.3μg·mL -1 , the detection limit is 6.20ng·mL -1 .

[0110] This analytical method was used to detect CRP antigen in spiked serum samples. Samples were taken, and the CL values ​​before and after the spiked samples were determined, and the concentrations were substituted into the linear equation. The results are shown in Table 1.

[0111] Table 1 CRP antigen detection results in spiked serum samples

[0112]

[0113] As can be seen in Table 1, the analytical method showed good accuracy and precision in detecting CRP antigen in spiked serum samples. The recovery rate ranged from 94.3% to 103.2%, indicating that the method was able to effectively detect CRP antigen from the samples and the test results were very close to the actual values. The relative standard deviation (RSD) was less than 5.6%, which further confirmed the high repeatability and stability of the method. These results together demonstrated the reliability and applicability of the developed method, making it a powerful tool for detecting CRP antigen.

[0114] 4. Anti-interference test

[0115] In order to evaluate the effect of other interfering substances in serum samples on CRP antigen detection, the concentration of 200 ng mL -1 The CRP-Ag standards were 1000 ng·mL -1 The antigen standards (IL-6, cTnT, P24, tPAI·C, SAA, PCT) were mixed in a volume of 1:1 to obtain an antigen mixture, and 5 μL of the antigen mixture was added to each 96-well plate. Then 20 μL of luminescent microspheres-CRP-coated antibody complex and 20 μL of biotin-CRP labeled antibody complex were added. The plate was incubated at 37°C for 5 minutes and gently stirred on a shaking platform. Finally, 40 μL of streptavidin-coated photosensitive microspheres were added to the reaction mixture in all wells and incubated for another 5 minutes at 37°C in the dark. The CL intensity was then measured on a LICA reader. The results are as follows. Fig.14 As shown, the CL value of this detection method does not change significantly when other antigens are present at the same time, indicating that this detection method has good anti-interference ability.

[0116] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A detection system for C-reactive protein, characterized in that: It includes luminescent microsphere-CRP coated antibody complex, biotin-CRP labeled antibody complex and streptavidin coated photosensitive microsphere.

2. The detection system according to claim 1, characterized in that The preparation method of the luminescent microsphere-CRP coated antibody complex comprises: mixing the activated luminescent microsphere with the C-reactive protein coated antibody at a mass ratio of 2:5, and coupling to form the luminescent microsphere-CRP coated antibody complex.

3. The detection system according to claim 1, characterized in that The preparation method of the biotin-CRP labeled antibody complex comprises: concentrating the CRP labeled antibody and removing impurities, then mixing the CRP labeled antibody solution and the biotin solution at a volume concentration ratio of 1:50, and incubating to obtain the biotin-CRP labeled antibody; The concentration of the biotin solution is 10 mmol·L -1 ; The concentration of the CRP coated antibody solution is 500ug·mL -1 ; The concentration of the CRP labeled antibody solution is 2.5 mg·mL -1 ; The concentration and impurity removal treatment method is dialysis or ultrafiltration tube treatment.

4. The detection system according to claim 1, characterized in that The volume ratio of the luminescent microsphere-CRP coated antibody complex, the biotin-CRP labeled antibody complex and the streptavidin coated photosensitive microsphere is 1:1:

2.

5. A method for detecting C-reactive protein for purposes other than disease diagnosis or treatment, characterized in that: The following steps are involved: (1) mixing the sample to be tested with the luminescent microsphere-CRP coated antibody complex and the biotin-CRP labeled antibody complex for the first incubation; (2) After the incubation is completed, streptavidin-coated photosensitive microspheres are added for a second incubation; (3) Determining the content of C-reactive protein in the sample based on the photochemiluminescence signal value after the second incubation.

6. The detection method according to claim 5, characterized in that: The volume ratio of the sample to be tested to the luminescent microsphere-CRP coated antibody complex is 1:

4.

7. The detection method according to claim 5, characterized in that: The conditions for measuring the CL value are: laser power is 110 mW, and irradiation time is 1200 ms.

8. The detection method according to claim 5, characterized in that: The conditions of the first incubation and the second incubation were: incubation at 37° C. for 5 min.

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