A photochemical chemiluminescence detection reagent, a preparation method thereof and a procalcitonin detection method
By using a method of conjugating procalcitonin antibodies to receptors and donor microspheres modified with surface carboxyl groups, the problems of low sensitivity and poor stability of existing photo-induced chemiluminescence detection methods have been solved, achieving more efficient and stable procalcitonin detection.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- GUANGDONG PHARMA UNIV
- Filing Date
- 2024-06-17
- Publication Date
- 2026-06-05
Smart Images
Figure CN118746685B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of photo-induced chemiluminescence detection technology, and in particular to a photo-induced chemiluminescence detection reagent and its preparation method, as well as a method for detecting procalcitonin. Background Technology
[0002] Procalcitonin (PCT) is a glycoprotein composed of 116 non-hormonally active amino acids, a precursor peptide of calcitonin. Under normal physiological conditions, it is produced by parathyroid C cells, hence it is also known as thyroid calcitonin. Its half-life is 25–30 hours. PCT can also be secreted by different types of cells in many organs in response to pro-inflammatory stimuli, particularly bacterial stimuli. For example, plasma levels of PCT increase in severe bacterial, fungal, or parasitic infections, as well as in sepsis and multiple organ failure.
[0003] Currently, there are various methods for detecting procalcitonin, such as radioimmunoassay, colloidal gold colorimetry, and photochemiluminescence immunoassay. However, current methods still have certain limitations. For example, radioimmunoassay has high sensitivity, but it is time-consuming and cannot distinguish between free and bound procalcitonin. Colloidal gold colorimetry cannot determine the specific value of procalcitonin; it can only visually compare the band colors to determine the range of procalcitonin levels, making it only a semi-quantitative method and unable to accurately detect the concentration of procalcitonin in the sample.
[0004] Photo-induced chemiluminescence (PET) is a homogeneous, wash-free chemiluminescence analysis technique based on nanoscale polymer particles and excited by light. Compared to traditional immunoassay methods, it offers advantages such as speed, homogeneity (wash-free), high sensitivity, and ease of operation. However, existing PET methods suffer from limitations including low detection sensitivity, poor precision, and poor stability.
[0005] For example, CN104597236A discloses a rapid detection method for procalcitonin and a corresponding detection kit. The detection method is as follows: (1) Mixing nanoscale donor microspheres with dye inside, receptor microspheres coated with active molecules, biotin-labeled active molecules, and the sample to be tested to obtain a homogenate; (2) Incubating the homogenate at a low temperature, the components with immunogenic properties react to obtain a conjugate; (3) Irradiating the conjugate with excitation light, the conjugate emits light, and the detection value is obtained by measuring with a photon counter. This method uses receptor microspheres and donor microspheres that are modified with aldehyde groups. However, aldehyde groups are more prone to oxidation or reduction reactions, which leads to changes in the performance of the microspheres. Moreover, aldehyde groups have poor flexibility and specificity when connecting other molecules or groups, and cannot accurately bind to the target molecule. In addition, this method requires reagents such as biotin and streptavidin to complete the detection, and the coupling steps are complex and time-consuming.
[0006] In view of this, the present invention is hereby proposed. Summary of the Invention
[0007] One objective of this invention is to provide a photocatalytic chemiluminescence detection reagent. The photocatalytic chemiluminescence detection reagent comprises acceptor microspheres and donor microspheres; wherein the acceptor microspheres are surface-modified with carboxyl groups, and the carboxyl groups are activated by sulfo-NHS / EDC and coupled with a procalcitonin antibody; and the donor microspheres are surface-modified with carboxyl groups, and the carboxyl groups are activated by sulfo-NHS / EDC and coupled with a procalcitonin antibody. The photocatalytic chemiluminescence detection reagent of this invention exhibits higher stability under various environments, can bind more precisely to target molecules, and achieve more efficient detection and recognition, enabling the microspheres to more accurately capture and immobilize target molecules, thereby facilitating subsequent luminescent reactions.
[0008] A second objective of this invention is to provide a method for preparing the aforementioned photo-induced chemiluminescence detection reagent. The preparation method sequentially includes: performing a first washing, activating the carboxyl groups, a second washing, conjugating an antibody, blocking unbound sites, a third washing, and mixing on carboxyl-modified acceptor microspheres and carboxyl-modified donor microspheres, thereby preparing a photo-induced chemiluminescence detection reagent containing acceptor microspheres and donor microspheres.
[0009] A third objective of this invention is to provide an application of the aforementioned photo-induced chemiluminescence detection reagent in the detection of procalcitonin.
[0010] The fourth objective of this invention is to provide a method for detecting procalcitonin. This detection method has high sensitivity and a wide measurement range.
[0011] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:
[0012] In a first aspect, the present invention provides a photo-induced chemiluminescence detection reagent, the photo-induced chemiluminescence detection reagent comprising acceptor microspheres and donor microspheres;
[0013] The receptor microspheres are surface-modified with carboxyl groups, and the carboxyl groups are activated by sulfo-NHS / EDC and coupled with procalcitonin antibody; the donor microspheres are surface-modified with carboxyl groups, and the carboxyl groups are activated by sulfo-NHS / EDC and coupled with procalcitonin antibody.
[0014] In this invention, the photoluminescence detection reagent contains acceptor microspheres and donor microspheres. Specific antibodies (procalcitonin antibodies) are coupled to the surfaces of the photosensitive microparticles and luminescent microparticles, respectively. When used for procalcitonin detection, the two microparticles are brought close together. The photosensitive microparticles, upon receiving excitation light, release singlet oxygen to form an "oxygen bridge" that transfers energy, causing the luminescent microparticles to emit a light signal. Detection is performed using a BPCL photoluminescence analyzer, which has advantages such as speed, homogeneity (wash-free), high sensitivity, and ease of operation.
[0015] In this invention, the selection of surface-carboxyl-modified acceptor microspheres and surface-carboxyl-modified donor microspheres for mixing offers the following advantages: 1. High stability: Carboxyl groups are chemically stable and less prone to unnecessary chemical reactions. This means that carboxyl-modified microspheres exhibit higher stability under various conditions, ensuring stable performance during storage and use. 2. Strong binding force: Compared to other groups, carboxyl groups offer greater flexibility and specificity in binding to other molecules, enabling carboxyl-modified microspheres to bind more precisely to target molecules, achieving more efficient detection and identification.
[0016] In this invention, the carboxyl groups are activated using a sulfo-NHS / EDC activation method. This step imbues the surfaces of both the acceptor and donor microspheres with specific functional groups. These functional groups can interact with target molecules or biomolecules, leading to antigen-antibody binding. This interaction is a crucial step in photochemiluminescence experiments, enabling the microspheres to more accurately and stably capture and immobilize procalcitonin target molecules, thereby facilitating subsequent luminescent reactions and accelerating the detection process, further improving the sensitivity and range of the detection method.
[0017] Preferably, the volume ratio of the acceptor microspheres to the donor microspheres is 1:(1-4), for example, it can be 1:1, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2, 1:2.2, 1:2.4, 1:2.6, 1:2.8, 1:3.2, 1:3.2, 1:3.4, 1:3.6, 1:3.8, 1:4, etc., preferably 1:2.
[0018] In this invention, when the photo-induced chemiluminescence detection reagent is mixed with a certain ratio of acceptor microspheres and donor microspheres, it is found that as the ratio of acceptor microspheres to donor microspheres increases, the chemiluminescence signal value first increases and then decreases; in particular, when the ratio of the two reaches 1:2, the chemiluminescence signal value is the largest.
[0019] Preferably, the concentration of the receptor microspheres in the photo-induced chemiluminescence detection reagent is 20–400 μg / mL, for example, it can be 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 45 μg / mL, 50 μg / mL, 55 μg / mL, 60 μg / mL, 65 μg / mL, 70 μg / mL, 75 μg / mL, 80 μg / mL, 85 μg / mL, 90 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 350 μg / mL, 400 μg / mL, etc., and preferably 50 μg / mL.
[0020] Preferably, the concentration of the donor microspheres in the photo-induced chemiluminescence detection reagent is 20–400 μg / mL, for example, it can be 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 45 μg / mL, 50 μg / mL, 55 μg / mL, 60 μg / mL, 65 μg / mL, 70 μg / mL, 75 μg / mL, 80 μg / mL, 85 μg / mL, 90 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 350 μg / mL, 400 μg / mL, etc., and preferably 50 μg / mL.
[0021] In this invention, the acceptor microspheres and donor microspheres in the detection reagent are diluted to a certain concentration using a diluent. As the dilution factor of the donor microspheres and acceptor microspheres increases, the chemiluminescence signal value first increases and then decreases. In particular, the chemiluminescence signal value is the largest when the concentration of the acceptor microspheres is 50 μg / mL and the concentration of the donor microspheres is 50 μg / mL.
[0022] Preferably, the solvent of the photo-induced chemiluminescence detection reagent is a homogeneous luminescence diluent (e.g., homogeneous luminescence diluent with catalog number HI0101A).
[0023] In this invention, the parameters of the receptor microspheres are as follows:
[0024] The receptor microspheres are polystyrene microspheres containing luminescent dyes; the receptor microspheres appear as a milky white liquid (e.g., Figure 1As shown), it appears red under ultraviolet light (as shown). Figure 2 (As shown).
[0025] The solid content of the receptor microspheres is 0.9% to 1.1%, for example, it can be 0.9%, 0.95%, 1.0%, 1.05%, 1.1%, etc.; and the storage solvent is pure water.
[0026] The particle size of the receptor microspheres is 180-220 nm, for example, it can be 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, etc.
[0027] The surface carboxyl content of the receptor microspheres is 80–120 μmol / g, for example, it can be 80 μmol / g, 85 μmol / g, 90 μmol / g, 95 μmol / g, 100 μmol / g, 105 μmol / g, 110 μmol / g, 115 μmol / g, 120 μmol / g, etc.
[0028] In this invention, the parameters of the donor microspheres are as follows:
[0029] The donor microspheres are polystyrene microspheres containing photosensitive dyes; the donor microspheres appear as a blue liquid (e.g., Figure 1 As shown), it appears light blue under ultraviolet light (as shown). Figure 2 (As shown).
[0030] The donor microspheres have a solid content of 0.9% to 1.1%, for example, 0.9%, 0.95%, 1.0%, 1.05%, 1.1%, etc.; and the storage solvent is pure water.
[0031] The donor microspheres have a particle size of 180–220 nm, for example, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, etc.
[0032] The surface carboxyl content of the donor microspheres is 80–120 μmol / g, for example, it can be 80 μmol / g, 85 μmol / g, 90 μmol / g, 95 μmol / g, 100 μmol / g, 105 μmol / g, 110 μmol / g, 115 μmol / g, 120 μmol / g, etc.
[0033] In a second aspect, the present invention provides a method for preparing a photo-induced chemiluminescence detection reagent as described in the first aspect, the preparation method comprising the following steps:
[0034] (1) The carboxyl-modified acceptor microspheres and the carboxyl-modified donor microspheres were washed for the first time using an activation buffer.
[0035] (2) The carboxyl groups on the surface of the carboxyl-modified acceptor microspheres and the carboxyl-modified donor microspheres were activated in the labeling buffer using sulfo-NHS and EDC, respectively.
[0036] (3) The recipient microspheres after carboxyl activation and the donor microspheres after carboxyl activation were washed a second time using activation buffer.
[0037] (4) The receptor microspheres with activated carboxyl groups and the donor microspheres with activated carboxyl groups were mixed with procalcitonin antibody in the labeling buffer to perform antibody conjugation.
[0038] (5) Block the unbinding sites of the receptor microspheres and the donor microspheres of the antibody-coupled procalcitonin.
[0039] (6) The receptor microspheres and donor microspheres of procalcitonin antibody after blocking sites were washed a third time with microsphere washing buffer, and then mixed with microsphere preservation solution to obtain the photo-induced chemiluminescence detection reagent.
[0040] In this invention, the preparation method includes: first washing, activating the carboxyl groups, second washing, conjugating an antibody, blocking unbound sites, third washing, and mixing of carboxyl-modified receptor microspheres and carboxyl-modified donor microspheres, thereby preparing a photo-induced chemiluminescence detection reagent containing receptor and donor microspheres. The photo-induced chemiluminescence detection reagent prepared by this method has the advantages of stable detection results, high accuracy, high sensitivity, good precision, and a wide detection range. Furthermore, the activation step of the preparation method enables the microspheres to more accurately and stably capture and immobilize the procalcitonin target molecule, thereby facilitating the subsequent luminescent reaction. The conjugation step is simpler, less time-consuming, and does not require reagents such as NaBH3CN.
[0041] Preferably, in step (1), the first cleaning specifically includes the following steps:
[0042] The carboxyl-modified acceptor microspheres and carboxyl-modified donor microspheres were mixed with activation buffer and centrifuged for the first time, and the supernatant was discarded. They were then mixed with activation buffer and centrifuged for the second time, and the supernatant was discarded to obtain the washed acceptor microspheres and the washed donor microspheres.
[0043] More specifically, in step (1), the first cleaning specifically includes the following steps:
[0044] (1-1) Washing of carboxyl-modified receptor microspheres: Mix the carboxyl-modified receptor microspheres with activation buffer, perform a first centrifugation, and discard the supernatant; add activation buffer, mix, perform a second centrifugation, and discard the supernatant to obtain the washed carboxyl-modified receptor microspheres.
[0045] (1-2) Washing of carboxyl-modified donor microspheres: Mix carboxyl-modified donor microspheres with activation buffer, perform a first centrifugation, and discard the supernatant; add activation buffer, mix, perform a second centrifugation, and discard the supernatant to obtain the washed donor microspheres.
[0046] Preferably, in step (1), the activation buffer is a MES activation buffer.
[0047] Preferably, in step (1), during the first and second centrifugation processes, the temperature is 10-20℃, for example, 10℃, 12℃, 14℃, 16℃, 18℃, 20℃, etc., the rotation speed is 12000-16000rpm, for example, 12000rpm, 13000rpm, 14000rpm, 15000rpm, 16000rpm, etc., and the time is 20-30min, for example, 20min, 22min, 24min, 26min, 28min, 30min, etc.
[0048] Preferably, in step (2), the activation specifically includes the following steps:
[0049] Labeling buffer was added to the cleaned acceptor microspheres and the cleaned donor microspheres respectively and mixed well. Then, EDC solution and sulfo-NHS solution were added sequentially and mixed well. Finally, activation reactions were carried out to obtain activated acceptor microspheres and activated donor microspheres.
[0050] More specifically, in step (2), the activation specifically includes the following steps:
[0051] (2-1) Activation of receptor microspheres: Add labeling buffer to the cleaned carboxyl-modified receptor microspheres and mix well, then add EDC solution and mix well, then add sulfo-NHS solution and mix well, and finally carry out the activation reaction to obtain the activated carboxyl receptor microspheres.
[0052] (2-2) Activation of donor microspheres: Add labeling buffer to the cleaned carboxyl-modified donor microspheres and mix well, then add EDC solution and mix well, then add sulfo-NHS solution and mix well, and finally carry out the activation reaction to obtain the activated carboxyl donor microspheres.
[0053] Preferably, in step (2), the labeling buffer is borate buffer.
[0054] Preferably, in step (2), the mass ratio of the receptor microspheres, labeling buffer, EDC and sulfo-NHS is 1:(2-4):(0.05-0.1):(0.5-0.7);
[0055] Among them, "2 to 4" can be, for example, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.5, 4, etc.;
[0056] Among them, "0.05~0.1" can be, for example, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, etc.;
[0057] Among them, "0.5~0.7" can be, for example, 0.5, 0.52, 0.55, 0.58, 0.6, 0.62, 0.65, 0.7, etc.
[0058] Preferably, in step (2), the mass ratio of the donor microspheres, labeling buffer, EDC and sulfo-NHS is 1:(2-4):(0.05-0.1):(0.5-0.7);
[0059] Among them, "2 to 4" can be, for example, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.5, 4, etc.;
[0060] Among them, "0.05~0.1" can be, for example, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, etc.;
[0061] Among them, "0.5~0.7" can be, for example, 0.5, 0.52, 0.55, 0.58, 0.6, 0.62, 0.65, 0.7, etc.
[0062] Preferably, in step (2), the activation temperature is 20-30°C, for example, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, etc., the rotation speed is 10-100 r / min, for example, 10 r / min, 20 r / min, 30 r / min, 40 r / min, 50 r / min, 60 r / min, 70 r / min, 80 r / min, 90 r / min, 100 r / min, etc., and the time is 10-30 min, for example, 10 min, 15 min, 20 min, 25 min, 30 min, etc.
[0063] Preferably, in step (3), the second cleaning specifically includes the following steps:
[0064] The recipient microspheres and donor microspheres after carboxyl activation were centrifuged for the first time and the supernatant was discarded. They were then mixed with activation buffer and centrifuged for the second time and the supernatant was discarded. Finally, they were mixed with ultrapure water and centrifuged for the third time and the supernatant was discarded.
[0065] More specifically, in step (3), the second cleaning specifically includes the following steps:
[0066] (3-1) Cleaning of the activator in the receptor microspheres: The receptor microspheres after activation of carboxyl groups were centrifuged for the first time and the supernatant was discarded; then they were mixed with activation buffer and centrifuged for the second time and the supernatant was discarded; finally, they were mixed with ultrapure water and centrifuged for the third time and the supernatant was discarded to obtain the cleaned activated receptor microspheres.
[0067] (3-2) Cleaning of the activator in the donor microspheres: The donor microspheres after activating the carboxyl groups were centrifuged for the first time and the supernatant was discarded; then they were mixed with the activation buffer and centrifuged for the second time and the supernatant was discarded; finally, they were mixed with ultrapure water and centrifuged for the third time and the supernatant was discarded to obtain the cleaned activated donor microspheres.
[0068] Preferably, in step (3), during the first centrifugation, the second centrifugation and the third centrifugation, the temperature is 10-20℃, for example, 10℃, 12℃, 14℃, 16℃, 18℃, 20℃, etc., the rotation speed is 12000-16000rpm, for example, 12000rpm, 13000rpm, 14000rpm, 15000rpm, 16000rpm, etc., and the time is 20-30min, for example, 20min, 22min, 24min, 26min, 28min, 30min, etc.
[0069] Preferably, in step (4), the conjugation of the antibody specifically includes the following steps:
[0070] Labeling buffer was added to the activated carboxyl group recipient microspheres and the activated carboxyl group donor microspheres, respectively, followed by the addition of procalcitonin antibody. Finally, the microspheres were incubated to obtain the procalcitonin antibody-coupled receptor microspheres and the procalcitonin antibody-coupled donor microspheres.
[0071] More specifically, in step (4), the conjugation of the antibody specifically includes the following steps:
[0072] (4-1) Receptor microspheres coupled with procalcitonin antibody: After washing and activating the receptor microspheres, add labeling buffer and mix well, then add procalcitonin antibody and mix well, and incubate to obtain receptor microspheres coupled with procalcitonin antibody.
[0073] (4-2) Donor microspheres conjugated with procalcitonin antibody: After washing and activating the donor microspheres, add labeling buffer and mix well, then add procalcitonin antibody and mix well, and incubate to obtain donor microspheres conjugated with procalcitonin antibody.
[0074] Preferably, in step (4), the mass ratio of the activated carboxyl receptor microspheres to the procalcitonin antibody is 1:(0.005-0.05), for example, it can be 1:0.005, 1:0.006, 1:0.008, 1:0.01, 1:0.02, 1:0.03, 1:0.04, 1:0.05, etc.
[0075] Preferably, in step (4), the mass ratio of the activated carboxyl donor microspheres to the procalcitonin antibody is 1:(0.005-0.05), for example, it can be 1:0.005, 1:0.006, 1:0.008, 1:0.01, 1:0.02, 1:0.03, 1:0.04, 1:0.05, etc.
[0076] Preferably, in step (4), the incubation temperature is 20-30°C, for example, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, etc., the rotation speed is 10-100 r / min, for example, 10 r / min, 20 r / min, 30 r / min, 40 r / min, 50 r / min, 60 r / min, 70 r / min, 80 r / min, 90 r / min, 100 r / min, etc., and the time is 1-3 h, for example, 1 h, 1.2 h, 1.4 h, 1.6 h, 1.8 h, 2 h, 2.2 h, 2.4 h, 2.6 h, 2.8 h, 3 h, etc.
[0077] Preferably, in step (5), the closing specifically includes the following steps:
[0078] Blocking solution was added to both the receptor microspheres and the donor microspheres of the procalcitonin antibody-conjugated antibody to carry out a blocking reaction, thereby blocking the unbound sites.
[0079] More specifically, in step (5), the closure specifically includes the following steps:
[0080] (5-1) Blocking of unbound sites on receptor microspheres: Blocking solution was added to receptor microspheres conjugated with procalcitonin antibody to carry out the blocking reaction and obtain blocked receptor microspheres.
[0081] (5-2) Blocking of unbound sites on donor microspheres: Blocking solution was added to donor microspheres conjugated with procalcitonin antibody to carry out the blocking reaction and obtain blocked donor microspheres.
[0082] Preferably, in step (5), the sealing liquid used is Western rapid sealing liquid.
[0083] Preferably, in step (5), the mass ratio of the receptor microspheres and blocking solution of the procalcitonin antibody conjugated is 1:(0.05-0.2), for example, it can be 1:0.05, 1:0.06, 1:0.08, 1:0.1, 1:0.12, 1:0.14, 1:0.16, 1:0.18, 1:0.2, etc.
[0084] Preferably, in step (5), the mass ratio of the donor microspheres and blocking solution of the conjugated procalcitonin antibody is 1:(0.05-0.2), for example, it can be 1:0.05, 1:0.06, 1:0.08, 1:0.1, 1:0.12, 1:0.14, 1:0.16, 1:0.18, 1:0.2, etc.
[0085] Preferably, in step (5), the sealing temperature is 35-40℃, for example, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, etc., the rotation speed is 10-100r / min, for example, 10r / min, 20r / min, 30r / min, 40r / min, 50r / min, 60r / min, 70r / min, 80r / min, 90r / min, etc., and the time is 15-30min, for example, 15min, 16min, 18min, 20min, 25min, 28min, 30min, etc.
[0086] Preferably, in step (6), the third cleaning specifically includes the following steps:
[0087] The receptor microspheres and donor microspheres conjugated with procalcitonin antibody after the blocking site were centrifuged for the first time and the supernatant was discarded. They were then mixed with microsphere washing buffer and centrifuged for the second time and the supernatant was discarded. They were then mixed with microsphere preservation solution to obtain diluents for the receptor microspheres and donor microspheres.
[0088] More specifically, the third cleaning includes the following steps:
[0089] (6-1) Third washing of receptor microspheres: The sealed receptor microspheres were centrifuged for the first time and the supernatant was discarded; then they were mixed with microsphere washing buffer and centrifuged for the second time and the supernatant was discarded; then they were mixed with microsphere preservation solution to obtain a diluted solution of receptor microspheres.
[0090] (6-2) Third washing of donor microspheres: The sealed donor microspheres were centrifuged for the first time and the supernatant was discarded; then they were mixed with microsphere washing buffer and centrifuged for the second time and the supernatant was discarded; then they were mixed with microsphere preservation solution to obtain a diluted solution of donor microspheres.
[0091] Preferably, in step (6), the microsphere washing buffer is PBS buffer.
[0092] Preferably, in step (6), the microsphere preservation solution is a homogeneous luminescent diluent (e.g., a homogeneous luminescent diluent with catalog number HI0101A).
[0093] Preferably, in step (6), during the first and second centrifugation processes, the temperature is 10-20℃, for example, 10℃, 12℃, 14℃, 16℃, 18℃, 20℃, etc., the rotation speed is 12000-16000rpm, for example, 12000rpm, 13000rpm, 14000rpm, 15000rpm, 16000rpm, etc., and the time is 20-30min, for example, 20min, 22min, 24min, 26min, 28min, 30min, etc.
[0094] Preferably, in step (7), the volume ratio of the diluent for the recipient microspheres to the diluent for the donor microspheres is 1:(1-4), for example, it can be 1:1, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2, 1:2.2, 1:2.4, 1:2.6, 1:2.8, 1:3.2, 1:3.2, 1:3.4, 1:3.6, 1:3.8, 1:4, etc., preferably 1:2.
[0095] Thirdly, the present invention provides an application of the photo-induced chemiluminescence detection reagent as described in the first aspect in the detection of procalcitonin.
[0096] Fourthly, the present invention provides a method for detecting procalcitonin, the method comprising the following steps:
[0097] The standard of procalcitonin was diluted with buffer solution to prepare standard solutions of different concentrations.
[0098] Standard solutions of different concentrations were mixed with the photo-induced chemiluminescence detection reagent and incubated. The photo-induced chemiluminescence signal values of the standards at different concentrations were then measured using a BPCL photo-induced chemiluminescence analyzer to establish a standard curve.
[0099] The test sample containing procalcitonin was mixed with the photo-induced chemiluminescence detection reagent and incubated. The photo-induced chemiluminescence signal value of the test sample was then detected using a BPCL photo-induced chemiluminescence analyzer and substituted into the standard curve to calculate the concentration of procalcitonin in the test sample.
[0100] Preferably, the buffer solution is PBS buffer.
[0101] Preferably, the concentration of the standard solution is 0.084 to 4.2 ng / mL, for example, it can be 0.084 ng / mL, 0.10 ng / mL, 0.21 ng / mL, 0.42 ng / mL, 0.84 ng / mL, 1.05 ng / mL, 2.1 ng / mL, 4.2 ng / mL, etc.
[0102] Preferably, the incubation temperature is 15 to 37°C, for example, it can be 15°C, 16°C, 18°C, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 33°C, 35°C, 37°C, etc., and preferably 37°C.
[0103] Preferably, the incubation time is 15s to 10min, for example, it can be 15s, 20s, 25s, 30s, 35s, 40s, 45s, 50s, 55s, 1min, 2min, 4min, 6min, 8min, 10min, etc., and preferably 10min.
[0104] Compared with the prior art, the present invention has the following beneficial effects:
[0105] (1) The photo-induced chemiluminescence detection reagent of the present invention has higher stability in various environments, can bind to target molecules more accurately, and achieve more efficient detection and identification, so that the microspheres can capture and fix target molecules more accurately, thereby carrying out subsequent luminescent reactions.
[0106] (2) The photo-induced chemiluminescence detection reagent prepared by the method has the advantages of stable detection results, high accuracy, high sensitivity, good precision, and wide detection range. Moreover, the preparation method is simpler, takes less time, and does not require reagents such as NaBH3CN. Attached Figure Description
[0107] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.
[0108] Figure 1 This is a schematic diagram showing the appearance of the acceptor microsphere (right) and donor microsphere (left) described in this invention.
[0109] Figure 2 This is a schematic diagram of the appearance of the acceptor microspheres (right) and donor microspheres (left) described in this invention under ultraviolet light.
[0110] Figure 3 The graph shows the relationship between the concentrations of acceptor and donor microspheres in the photo-induced chemiluminescence detection reagent provided in Example 1 and the luminescence signal value.
[0111] Figure 4 This is a graph showing the relationship between the mixing ratio of acceptor microspheres and donor microspheres and the luminescence signal value in the photo-induced chemiluminescence detection reagent provided in Example 2.
[0112] Figure 5 The graph shows the relationship between the amount of PCT-Ag standard provided in Example 3 and the luminescence signal value.
[0113] Figure 6 The graph shows the relationship between incubation temperature and photochemiluminescence signal value in PCT-Ag detection provided in Example 4.
[0114] Figure 7 The graph shows the relationship between incubation time and photochemiluminescence signal value in the PCT-Ag detection provided in Example 5.
[0115] Figure 8 The standard curve is shown for the detection method of procalcitonin provided in Example 1. Detailed Implementation
[0116] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.
[0117] It should be noted that specific details are set forth in the following description to provide a full understanding of the invention. However, the invention can be practiced in many ways other than those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.
[0118] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0119] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.
[0120] The sources and parameters of the recipient and donor microspheres in the following examples are shown below:
[0121] Receptor microspheres: Polystyrene microspheres containing luminescent dyes, purchased from Weidu Biotechnology Co., Ltd.; product code 67700001, batch number 2230009A, specification 1mL; the receptor microspheres appear as a milky white liquid (e.g. Figure 1 As shown), it appears red under ultraviolet light (as shown). Figure 2 (as shown); the solid content is 1.1%, the average particle size is 220 nm, and the surface carboxyl content is 85 μmol / g.
[0122] Donor microspheres: Polystyrene microspheres containing photosensitive dyes, purchased from Weidu Biotechnology Co., Ltd.; product code 67500001, batch number 2310209A, specification 1mL; the appearance of the donor microspheres is a blue liquid (e.g., Figure 1 As shown), it appears light blue under ultraviolet light (as shown). Figure 2 (as shown); the solid content is 1.1%, the average particle size is 195 nm, and the surface carboxyl content is 100 μmol / g.
[0123] Activation buffer: MES activation buffer, pH 6.0.
[0124] Labeling buffer: borate buffer, 40 mM, pH 7.8.
[0125] Sealing fluid: QuickBlock TM Western blotting fluid, 100 μL.
[0126] Microsphere washing buffer: PBS buffer, pH 7.4.
[0127] Microsphere preservation solution: homogeneous luminescent diluent, purchased from Weidu Biotechnology, catalog number HI0101A.
[0128] Preparation Example 1
[0129] This preparation example provides a receptor microsphere conjugated with procalcitonin antibody, which is prepared by the following steps:
[0130] (1) Cleaning of receptor microspheres
[0131] Take 0.1 mL of receptor microspheres into a 2 mL centrifuge tube containing 1 mL of pH 6.0 MES activation buffer, sonicate to mix, centrifuge at 15℃ and 14000 rpm for 25 min, discard the supernatant, add 1 mL of activation buffer, sonicate to disperse evenly, centrifuge at 15℃ and 14000 rpm for 25 min, and discard the supernatant.
[0132] (2) Surface activation of receptor microspheres
[0133] Add 1 mL of microsphere labeling buffer to the centrifuge tube from step (1), sonicate to disperse evenly, then add 7 μL of EDC solution (10 mg / mL), vortex to mix, then add 66 μL of sulfo-NHS solution (10 mg / mL), sonicate to mix; place the centrifuge tube in a constant temperature shaker and activate it for 20 min at 25 °C and 60 r / min in the dark.
[0134] (3) Cleaning of the receptor microspheres with activator
[0135] Place the centrifuge tube from step (2) into a high-speed refrigerated centrifuge and centrifuge at 15°C and 14,000 rpm for 25 min. Discard the supernatant, resuspend the microspheres in 1 mL of activation buffer by sonication, and continue centrifuging at 15°C and 14,000 rpm for 25 min. Discard the supernatant, resuspend the microspheres in 1 mL of pure water by sonication, and repeat the washing step once.
[0136] (4) Antibodies conjugated to receptor microspheres
[0137] Add 1 mL of labeling buffer to the centrifuge tube from step (3) and sonicate to disperse evenly; add 20 μg of PCT antibody to the sonicated microsphere solution, vortex to mix, and place the centrifuge tube in a constant temperature shaker and incubate at 25°C and 60 r / min for 2 h, avoiding light throughout the process.
[0138] (5) Blocking unbinding sites of receptor microspheres
[0139] Add 100 μL of rapid blocking solution to the centrifuge tube from step (4), mix well, and place the centrifuge tube in a constant temperature shaker at 37°C in the dark for 30 min.
[0140] (6) Wash thoroughly
[0141] Place the centrifuge tube from step (5) in a high-speed refrigerated centrifuge and centrifuge at 15°C and 14,000 rpm for 25 min. Discard the supernatant, add 1 mL of microsphere washing buffer, and repeat the centrifugation and washing three times. Finally, add 1 mL of microsphere preservation solution and sonicate to disperse evenly to obtain receptor microspheres coupled with procalcitonin antibody. Store at 2–8°C in the dark for later use.
[0142] Preparation Example 2
[0143] This preparation example provides a donor microsphere conjugated with procalcitonin antibody, which is prepared by the following steps:
[0144] (1) Cleaning of donor microspheres
[0145] Take 0.1 mL of donor microspheres into a 2 mL centrifuge tube containing 1 mL of pH 6.0 MES activation buffer, sonicate to mix, centrifuge at 15℃ and 14000 rpm for 25 min, discard the supernatant, add another 1 mL of activation buffer, sonicate to disperse evenly, centrifuge at 15℃ and 14000 rpm for 25 min, and discard the supernatant.
[0146] (2) Surface activation of donor microspheres
[0147] Add 1 mL of microsphere labeling buffer to the centrifuge tube from step (1), sonicate to disperse evenly, then add 7 μL of EDC solution (10 mg / mL), vortex to mix, then add 66 μL of sulfo-NHS solution (10 mg / mL), sonicate to mix; place the centrifuge tube in a constant temperature shaker and activate it for 20 min at 25 °C and 60 r / min in the dark.
[0148] (3) Cleaning of the activator for the donor microspheres
[0149] Place the centrifuge tube from step (2) into a high-speed refrigerated centrifuge and centrifuge at 15°C and 14,000 rpm for 25 min. Discard the supernatant, resuspend the microspheres in 1 mL of activation buffer by sonication, and continue centrifuging at 15°C and 14,000 rpm for 25 min. Discard the supernatant, resuspend the microspheres in 1 mL of pure water by sonication, and repeat the washing step once.
[0150] (4) Antibody conjugation to donor microspheres
[0151] Add 1 mL of labeling buffer to the centrifuge tube from step (3) and sonicate to disperse evenly; add 20 μg of PCT antibody to the sonicated microsphere solution, vortex to mix, and place the centrifuge tube in a constant temperature shaker and incubate at 25°C and 60 r / min for 2 h, avoiding light throughout the process.
[0152] (5) Closed unbound sites of donor microspheres
[0153] Add 100 μL of rapid blocking solution to the centrifuge tube from step (4), mix well, and place the centrifuge tube in a constant temperature shaker at 37°C in the dark for 30 min.
[0154] (6) Wash thoroughly
[0155] Place the centrifuge tube from step (5) in a high-speed refrigerated centrifuge and centrifuge at 15°C and 14,000 rpm for 25 min. Discard the supernatant, add 1 mL of microsphere washing buffer, and repeat the centrifugation and washing three times. Finally, add 1 mL of microsphere preservation solution and sonicate to disperse evenly to obtain donor microspheres coupled with procalcitonin antibody. Store at 2–8°C in the dark for later use.
[0156] Example 1
[0157] Concentration determination of acceptor and donor microspheres in photo-induced chemiluminescence assay reagent
[0158] Test method: The receptor microspheres of PCT monoclonal antibody conjugated provided in Preparation Example 1 and the donor microspheres of PCT monoclonal antibody conjugated provided in Preparation Example 2 were diluted to different factors. The two types of microspheres were then conjugated with PCT-Ag standard stepwise, and then tested by instrument to determine the optimal dilution factor.
[0159] Test results are as follows Figure 3 As shown, with increasing dilution factors of donor and acceptor microspheres, the chemiluminescence signal value first increases and then decreases. Considering minimizing reagent usage, the rate of change in the chemiluminescence signal value is greatest at an 8-fold dilution; therefore, the optimal dilution factor is considered to be 8-fold. Specifically, the concentration of the acceptor microspheres conjugated with PCT monoclonal antibody provided in Example 1 of the photo-induced chemiluminescence detection reagent is 50 μg / mL; and the concentration of the donor microspheres conjugated with PCT monoclonal antibody provided in Example 2 of the photo-induced chemiluminescence detection reagent is 50 μg / mL.
[0160] Example 2
[0161] Test of the mixing ratio of acceptor microspheres and donor microspheres in photo-induced chemiluminescence detection reagents
[0162] Test method: Take 20 μL of the receptor microspheres of the PCT monoclonal antibody conjugated provided in Preparation Example 1 into each white opaque 96-well plate, add 20 μL of 1.05 ng / mL PCT-Ag standard, and place in a constant temperature shaker at 37°C for 15 s to ensure thorough mixing. Immediately afterwards, add 20 μL, 30 μL, 40 μL, 60 μL, and 80 μL of the donor microspheres of the PCT monoclonal antibody conjugated in Preparation Example 2, respectively, i.e., the ratio of receptor microspheres to donor microspheres is 1:1, 2:3, 1:2, 1:3, and 1:4. Continue shaking at 37°C for 15 s. After shaking, use a BPCL photochemiluminescence analyzer to test the photochemiluminescence signal values of each photochemiluminescence detection reagent with different mixing ratios.
[0163] Test results are as follows Figure 4 As shown: Based on the fixed amount of acceptor microspheres (20 μL) and PCT-Ag standard (20 μL), different volumes of donor microspheres were added to investigate the optimal ratio of acceptor microspheres to donor microspheres. The results showed that as the ratio of donor microspheres to acceptor microspheres increased, the chemiluminescence signal value first increased and then decreased. When the ratio of the two was 1:2, the chemiluminescence signal value was the largest, and this ratio was considered to be the optimal ratio.
[0164] Example 3
[0165] Testing the most suitable amount of PCT-Ag standard
[0166] Test method: Take 20 μL of the receptor microspheres of PCT monoclonal antibody conjugated provided in Preparation Example 1 into a white opaque 96-well plate, and add 10 μL, 20 μL, 30 μL, 40 μL and 50 μL of 1.05 ng / mL PCT-Ag standard, respectively. Place the plates in a constant temperature shaker in the dark and shake at 37°C for 15 s to ensure thorough mixing. Immediately afterward, add 40 μL of the donor microspheres of PCT monoclonal antibody conjugated provided in Preparation Example 2 to each plate and continue shaking at 37°C for 15 s. Immediately after shaking, measure the photochemiluminescence signal value using a BPCL photochemiluminescence analyzer.
[0167] Test results are as follows Figure 5 As shown: Based on the amounts of fixed receptor microspheres (20 μL) and donor microspheres (40 μL), the optimal amount of PCT-Ag standard in the detection process was investigated. It can be seen that as the amount of PCT-Ag standard increases, the chemiluminescence signal value first increases and then decreases. However, when the amount of PCT-Ag standard is 40 μL and 50 μL, the difference in chemiluminescence signal value is not significant, indicating that the immune reaction is basically complete when the amount of PCT-Ag standard is 40 μL. Therefore, the optimal detection amount of the standard is 40 μL.
[0168] Example 4
[0169] Incubation temperature test
[0170] Test method: Take 20 μL of the acceptor microspheres of PCT monoclonal antibody provided in Preparation Example 1 into each white opaque 96-well plate, add 40 μL of 1.05 ng / mL PCT-Ag standard to each well, and place them in a constant temperature shaker in the dark. Shake for 15 s under different temperature conditions to ensure thorough mixing. Then immediately add 40 μL of the donor microspheres of PCT monoclonal antibody provided in Preparation Example 2 to each well, and continue shaking for 15 s under different temperature conditions. Immediately after shaking, use a BPCL photochemiluminescence analyzer to measure the photochemiluminescence signal value.
[0171] Test results are as follows Figure 6 As shown, the chemiluminescence signal value increases with both incubation temperature and incubation time. The signal value is highest at an incubation temperature of 37℃, followed by the signal value at an incubation temperature of 25℃.
[0172] Example 5
[0173] Incubation time test
[0174] Test method: Take 20 μL of the acceptor microspheres of PCT monoclonal antibody provided in Preparation Example 1 into each of the white opaque 96-well plates, add 40 μL of 1.05 ng / mL PCT-Ag standard to each well, and place them in a constant temperature shaker at 37°C for different times to ensure thorough mixing. Immediately afterwards, add 40 μL of the donor microspheres of PCT monoclonal antibody provided in Preparation Example 2 to each well, and continue to shake at 37°C for different times to ensure thorough mixing. Immediately afterwards, use a BPCL photochemiluminescence analyzer to measure the photochemiluminescence signal value.
[0175] Test results are as follows Figure 7 As shown, when the incubation time is 10 min, the chemiluminescence signal value of a higher PCT-Ag concentration does not change much, indicating that the immune response equilibrium has been reached after 10 min of incubation. Therefore, the optimal incubation temperature and time for PCT-Ag detection are 37℃ and 10 min, respectively.
[0176] Application Example 1
[0177] This application example provides a method for detecting procalcitonin, which includes the following steps:
[0178] (1) Establishment of the standard curve:
[0179] The PCT antigen standard was diluted with PBS buffer to 0.084, 0.105, 0.21, 1.05, 2.1 and 4.2 ng / mL, respectively.
[0180] Take 20 μL of the receptor microspheres of the PCT monoclonal antibody conjugated provided in Preparation Example 1 into a white opaque 96-well plate, add 40 μL of 1.05 ng / mL PCT-Ag standard to each well, place in a constant temperature shaker in the dark, and shake for 10 min at 37°C. Immediately afterwards, add 40 μL of the donor microspheres of the PCT monoclonal antibody conjugated provided in Preparation Example 2 to each well, and continue shaking for 10 min at 37°C. Immediately afterwards, use a BPCL photochemiluminescence analyzer to measure the photochemiluminescence signal value.
[0181] A standard curve was established with the chemiluminescence signal value as the ordinate and the PCT antigen concentration as the abscissa. The linear equation and the detection limit were then determined.
[0182] The results are as follows Figure 8 As shown, the linear equation is obtained as y = 408.48x + 246.72, R0 2 =0.9983, the linear range is 0.084-4.2 ng / mL, and the limit of detection is 0.02 ng / mL.
[0183] (2) Detection of the concentration of procalcitonin in the test sample:
[0184] The test sample containing procalcitonin was incubated according to the method used in the standard curve establishment process. Then, the photoluminescence signal value of the test sample was detected by the BPCL photoluminescence analyzer and substituted into the standard curve to calculate the concentration of procalcitonin in the test sample.
[0185] The specific tests are shown in Table 1:
[0186] Table 1
[0187]
[0188] As shown above, specific antibodies are coupled to the surfaces of two types of high-molecular-weight microparticles, namely photosensitive microparticles and luminescent microparticles. These antibodies then specifically bind to the antigen, bringing the two microparticles close together. When the photosensitive microparticles are irradiated with excitation light, they release singlet oxygen to form an "oxygen bridge," transferring energy and causing the luminescent microparticles to emit a light signal. This signal is then detected using a BPCL photochemical analyzer. The rapid detection method and kit for procalcitonin of this invention provide stable results, high accuracy, low cost, high sensitivity, good precision, and a wide detection range.
[0189] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. A photo-induced chemiluminescence detection reagent, characterized in that, The photo-induced chemiluminescence detection reagent comprises acceptor microspheres and donor microspheres; the volume ratio of the acceptor microspheres to the donor microspheres is 1:(1.8~2.2); The receptor microspheres are surface-modified with carboxyl groups, and the carboxyl groups are activated by sulfo-NHS / EDC and coupled with procalcitonin antibody; the particle size of the receptor microspheres is 180~220 nm, and the surface carboxyl group content of the receptor microspheres is 80~120 μmol / g; the concentration of receptor microspheres in the photo-induced chemiluminescence detection reagent is 40~60 μg / mL; The donor microspheres are carboxyl-modified microspheres with activated carboxyl groups via sulfo-NHS / EDC and coupled with procalcitonin antibody. The particle size of the donor microspheres is 180-220 nm, and the surface carboxyl content is 80-120 μmol / g. The concentration of the donor microspheres in the photo-induced chemiluminescence detection reagent is 40-60 μg / mL. The photo-induced chemiluminescence detection reagent is prepared by the following steps: (1) The carboxyl-modified acceptor microspheres and the carboxyl-modified donor microspheres were washed for the first time using activation buffer, respectively; (2) The carboxyl groups on the surface of the carboxyl-modified acceptor microspheres and the carboxyl-modified donor microspheres were activated in the labeling buffer using sulfo-NHS and EDC, respectively; (3) The recipient microspheres and donor microspheres after carboxyl activation were washed a second time using activation buffer. (4) The receptor microspheres with activated carboxyl groups and the donor microspheres with activated carboxyl groups were mixed with procalcitonin antibody in the labeling buffer to perform antibody conjugation. (5) Block the unbinding sites of the receptor microspheres and the donor microspheres of the antibody-coupled procalcitonin, respectively. (6) The receptor microspheres and donor microspheres coupled with procalcitonin antibody after blocking the site were washed a third time with microsphere washing buffer, and then mixed with microsphere preservation solution to obtain the photo-induced chemiluminescence detection reagent.
2. The photo-induced chemiluminescence detection reagent according to claim 1, characterized in that, The volume ratio of the acceptor microspheres to the donor microspheres is 1:
2.
3. The photo-induced chemiluminescence detection reagent according to claim 1, characterized in that, The concentration of the receptor microspheres in the photo-induced chemiluminescence detection reagent is 45~55 μg / mL.
4. The photo-induced chemiluminescence detection reagent according to claim 3, characterized in that, The concentration of the receptor microspheres in the photo-induced chemiluminescence detection reagent is 50 μg / mL.
5. The photo-induced chemiluminescence detection reagent according to claim 1, characterized in that, The concentration of donor microspheres in the photo-induced chemiluminescence detection reagent is 45~55 μg / mL.
6. The photo-induced chemiluminescence detection reagent according to claim 5, characterized in that, The concentration of donor microspheres in the photo-induced chemiluminescence detection reagent is 50 μg / mL.
7. A method for preparing a photo-induced chemiluminescence detection reagent according to any one of claims 1 to 6, characterized in that, The preparation method includes the following steps: (1) The carboxyl-modified acceptor microspheres and the carboxyl-modified donor microspheres were washed for the first time using activation buffer, respectively; (2) The carboxyl groups on the surface of the carboxyl-modified acceptor microspheres and the carboxyl-modified donor microspheres were activated in the labeling buffer using sulfo-NHS and EDC, respectively; (3) The recipient microspheres and donor microspheres after carboxyl activation were washed a second time using activation buffer. (4) The receptor microspheres with activated carboxyl groups and the donor microspheres with activated carboxyl groups were mixed with procalcitonin antibody in the labeling buffer to perform antibody conjugation. (5) Block the unbinding sites of the receptor microspheres and the donor microspheres of the antibody-coupled procalcitonin, respectively. (6) The receptor microspheres and donor microspheres coupled with procalcitonin antibody after blocking the site were washed a third time with microsphere washing buffer, and then mixed with microsphere preservation solution to obtain the photo-induced chemiluminescence detection reagent.
8. The method for preparing the photo-induced chemiluminescence detection reagent according to claim 7, characterized in that, In step (1), the first cleaning specifically includes the following steps: The carboxyl-modified acceptor microspheres and carboxyl-modified donor microspheres were mixed with activation buffer and centrifuged for the first time, and the supernatant was discarded. They were then mixed with activation buffer again and centrifuged for the second time, and the supernatant was discarded to obtain the washed acceptor microspheres and the washed donor microspheres.
9. The method for preparing the photo-induced chemiluminescence detection reagent according to claim 8, characterized in that, In step (1), the activation buffer is MES activation buffer.
10. The method for preparing the photo-induced chemiluminescence detection reagent according to claim 8, characterized in that, In step (1), during the first and second centrifugation processes, the temperature is 10~20℃, the rotation speed is 12000~16000rpm, and the time is 15~30min.
11. The method for preparing the photo-induced chemiluminescence detection reagent according to claim 7, characterized in that, In step (2), the activation specifically includes the following steps: Labeling buffer was added to the cleaned acceptor microspheres and the cleaned donor microspheres respectively and mixed well. Then, EDC solution and sulfo-NHS solution were added sequentially and mixed well. Finally, activation reactions were carried out to obtain activated acceptor microspheres and activated donor microspheres.
12. The method for preparing the photo-induced chemiluminescence detection reagent according to claim 11, characterized in that, In step (2), the labeling buffer is borate buffer.
13. The method for preparing the photo-induced chemiluminescence detection reagent according to claim 11, characterized in that, In step (2), the mass ratio of the receptor microspheres, labeling buffer, EDC and sulfo-NHS is 1:(2~4):(0.05~0.1):(0.5~0.7).
14. The method for preparing the photo-induced chemiluminescence detection reagent according to claim 11, characterized in that, In step (2), the mass ratio of the donor microspheres, labeling buffer, EDC and sulfo-NHS is 1:(2~4):(0.05~0.1):(0.5~0.7).
15. The method for preparing the photo-induced chemiluminescence detection reagent according to claim 11, characterized in that, In step (2), the activation temperature is 20~30℃, the rotation speed is 10~100 r / min, and the time is 10~30 min.
16. The method for preparing the photo-induced chemiluminescence detection reagent according to claim 7, characterized in that, In step (3), the second cleaning specifically includes the following steps: The recipient microspheres and donor microspheres after carboxyl activation were centrifuged for the first time and the supernatant was discarded. They were then mixed with activation buffer and centrifuged for the second time and the supernatant was discarded. Finally, they were mixed with ultrapure water and centrifuged for the third time and the supernatant was discarded.
17. The method for preparing the photo-induced chemiluminescence detection reagent according to claim 16, characterized in that, In step (3), during the first centrifugation, the second centrifugation and the third centrifugation, the temperature is 10~20℃, the rotation speed is 12000~16000 rpm, and the time is 20~30 min.
18. The method for preparing the photo-induced chemiluminescence detection reagent according to claim 7, characterized in that, In step (4), the conjugation of the antibody specifically includes the following steps: Labeling buffer was added to the activated carboxyl group recipient microspheres and the activated carboxyl group donor microspheres, respectively, followed by the addition of procalcitonin antibody. Finally, the microspheres were incubated to obtain the procalcitonin antibody-coupled receptor microspheres and the procalcitonin antibody-coupled donor microspheres.
19. The method for preparing the photo-induced chemiluminescence detection reagent according to claim 18, characterized in that, In step (4), the mass ratio of the activated carboxyl receptor microspheres to the procalcitonin antibody is 1:(0.005~0.05).
20. The method for preparing the photo-induced chemiluminescence detection reagent according to claim 18, characterized in that, In step (4), the mass ratio of the activated carboxyl donor microspheres to the procalcitonin antibody is 1:(0.005~0.05).
21. The method for preparing the photo-induced chemiluminescence detection reagent according to claim 18, characterized in that, In step (4), the incubation reaction is carried out at a temperature of 20-30°C, a rotation speed of 10-100 r / min, and a time of 1-3 h.
22. The method for preparing the photo-induced chemiluminescence detection reagent according to claim 7, characterized in that, In step (5), the closure specifically includes the following steps: Blocking solution was added to both the receptor microspheres and the donor microspheres of the procalcitonin antibody-conjugated antibody to carry out a blocking reaction, thereby blocking the unbound sites.
23. The method for preparing the photo-induced chemiluminescence detection reagent according to claim 22, characterized in that, In step (5), the sealing liquid used is Western rapid sealing liquid.
24. The method for preparing the photo-induced chemiluminescence detection reagent according to claim 22, characterized in that, In step (5), the mass ratio of the receptor microspheres and blocking solution of the conjugated procalcitonin antibody is 1:(0.05~0.2).
25. The method for preparing the photo-induced chemiluminescence detection reagent according to claim 22, characterized in that, In step (5), the mass ratio of the donor microspheres and blocking solution of the conjugated procalcitonin antibody is 1:(0.05~0.2).
26. The method for preparing the photo-induced chemiluminescence detection reagent according to claim 22, characterized in that, In step (5), the sealing temperature is 35~40℃, the rotation speed is 10~100 r / min, and the time is 15~30 min.
27. The method for preparing the photo-induced chemiluminescence detection reagent according to claim 7, characterized in that, In step (6), the third cleaning specifically includes the following steps: The receptor microspheres and donor microspheres conjugated with procalcitonin antibody after the blocking site were centrifuged for the first time and the supernatant was discarded. They were then mixed with microsphere washing buffer and centrifuged for the second time and the supernatant was discarded. Finally, they were mixed with microsphere preservation solution.
28. The method for preparing the photo-induced chemiluminescence detection reagent according to claim 27, characterized in that, In step (6), the microsphere washing buffer is PBS buffer.
29. The method for preparing the photo-induced chemiluminescence detection reagent according to claim 27, characterized in that, In step (6), the microsphere preservation solution is a homogeneous luminescent diluent.
30. The method for preparing the photo-induced chemiluminescence detection reagent according to claim 27, characterized in that, In step (6), during the first and second centrifugation processes, the temperature is 10~20℃, the rotation speed is 12000~16000rpm, and the time is 15~30 min.