A method and kit for immunoassay of a target substance using arterial blood

Abstract

The application discloses a method and a kit for immunodetection of target substances by using arterial blood, and belongs to the technical field of blood detection. The kit comprises at least: a first component containing luminescent microspheres combined with detection antibody 1 and a luminescent microsphere diluent, wherein the luminescent microsphere diluent contains 2-morpholinoethanesulfonic acid, KCl and NaHCO3; a second component containing biotin-labeled detection antibody 2 and a biotin diluent, wherein the biotin diluent contains dithiothreitol, CaCl2, adenine nucleoside and mouse anti-human hemoglobin antibody; and a third component containing photosensitive microspheres combined with streptomycin. The scheme can be used for immunodetection of target substances by using arterial blood, has excellent functional sensitivity and detection range, is not interfered by other substances in arterial blood, realizes rapid detection of all blood gas indexes and immunological indexes by using one tube of arterial blood, and has important significance for clinical blood detection.

Description

Technical Field

[0001] This invention relates to the field of blood testing technology, specifically to a method and kit for immunodetection of target substances using arterial blood. Background Technology

[0002] In chemiluminescence immunoassay, the determination of target analytes in human blood is always performed using venous blood, which has the following advantages compared to arterial blood: 1. Veins are more superficial, making puncture relatively simple and less risky, while arterial blood puncture is more difficult and carries a higher risk of complications such as hematoma and arterial spasm; 2. Venous blood flow is slower, and indicators such as oxygen and carbon dioxide content and pH are relatively stable, making it more suitable for immunoassay. In contrast, blood gas analysis requires monitoring of oxygen partial pressure (PO2) and carbon dioxide partial pressure (PCO2), necessitating arterial blood collection. When simultaneous immunoassay and blood gas analysis is required, separate arterial and venous blood samples must be collected, increasing the workload for medical staff and causing more pain and inconvenience for patients. In the resuscitation of critically ill patients, faster sample collection and testing shortens the treatment window, thus increasing the patient's chances of survival. Therefore, how to achieve rapid detection of all blood gas and immune indicators from a single tube of arterial blood has become a pressing issue that needs to be addressed in the daily work of emergency departments and ICUs, as well as other critical care and nursing departments.

[0003] Significant differences exist between arterial and venous blood in indicators such as partial pressure of oxygen and carbon dioxide, pH, and some electrolytes, which can affect the detection of some target antigens. This leads to substantial discrepancies between arterial and venous blood results for certain immunological markers. Therefore, current technology cannot directly use arterial blood for immunological marker detection. Furthermore, both arterial and venous blood may be affected by platelet aggregation and hemoglobin, which can interfere with the detection of the antigens being tested. This interference is particularly severe for homogeneous assay reagents, as they lack a separation and washing process throughout the assay.

[0004] Currently, the stability of commercially available liquid homogeneous assay reagents is generally poor, affecting the accuracy of clinical testing. To address this challenge, liquid reagents can be prepared into lyophilized reagents through vacuum freeze-drying. The principle involves rapid freezing at low temperatures, followed by sublimation under vacuum to remove moisture, ultimately yielding a solid reagent. Lyophilized homogeneous assay reagents can be transported and stored at room temperature while ensuring long-term stability. For example, Chinese patent application CN 114994304 A discloses a cTnI dry homogeneous chemiluminescence assay kit, but this kit can only be used for detecting venous serum and plasma samples, and cannot be applied to arterial blood samples. Summary of the Invention

[0005] In view of the technical problems existing in the background art, the present invention provides a method and kit for immunological detection of target substances using arterial blood, aiming to solve the technical problem that a single tube of arterial blood cannot be used for rapid detection of all blood gas indicators and immune indicators.

[0006] In a first aspect, the present invention provides a homogeneous detection kit for immunoassay of a target substance using arterial blood, comprising at least the following reagents:

[0007] The first component comprises luminescent microspheres bound to detection antibody 1 and a luminescent microsphere diluent, wherein the luminescent microsphere diluent contains 2-morpholinoethanesulfonic acid, KCl and NaHCO3;

[0008] The second component contains biotin-labeled detection antibody 2 and biotin diluent, and the biotin diluent contains dithiothreitol, CaCl2, adenine nucleoside and mouse anti-human hemoglobin antibody.

[0009] The third component comprises photosensitive microspheres conjugated with streptomycin and a photosensitive microsphere diluent.

[0010] It is understandable that in the above homogeneous detection kit, detection antibody 1 and detection antibody 2 are two different antibodies against the target substance. For example, when the target substance is troponin I (cTnI), detection antibody 1 and detection antibody 2 are two different anti-troponin I antibodies.

[0011] In the above homogeneous detection kit, the luminescent microspheres are polystyrene microspheres coated with europium as a luminescent substance, with a diameter of 200nm-300nm; the photosensitive microspheres are polystyrene microspheres coated with phthalocyanine as a photosensitive substance, with a diameter of 200nm-300nm.

[0012] Furthermore, in the first component described above, the concentration of the luminescent microspheres bound to the detection antibody 1 is 10 μg / ml-100 μg / ml, preferably 10 μg / ml-50 μg / ml, and more preferably 20 μg / ml-40 μg / ml.

[0013] Furthermore, in the second component described above, the concentration of the biotin-labeled detection antibody 2 is 0.1 μg / ml-10 μg / ml, preferably 0.5 μg / ml-5 μg / ml, and more preferably 1 μg / ml-3 μg / ml.

[0014] Furthermore, in the third component described above, the concentration of the photosensitive microspheres containing streptomycin is 20 μg / ml to 200 μg / ml, preferably 20 μg / ml to 100 μg / ml, and more preferably 20 μg / ml to 80 μg / ml.

[0015] Furthermore, the luminescent microsphere diluent contains 20mM-50mM 2-morpholinoethanesulfonic acid, 0.01%-0.2% KCl, and 0.1%-2% NaHCO3, while the biotin diluent contains 0.2mM-5mM dithiothreitol, 1mM-10mM CaCl2, 1mM-10mM adenine nucleoside, and 20μg / ml-100μg / ml mouse anti-human hemoglobin antibody. To address the interference issues in arterial blood testing, this invention adds 2-morpholinoethanesulfonic acid (which lowers the pH of arterial blood), dithiothreitol (a reducing agent that lowers dissolved oxygen levels to ensure that the partial pressures of oxygen and carbon dioxide in arterial blood are consistent with those in venous blood), and KCl to the diluents of each component. + Na + Ca 2+ Plasma is used to balance electrolytes in arterial blood, ensuring that the electrolyte concentration is close to that in venous blood. Adenosine and mouse anti-human hemoglobin antibodies are also added to inhibit platelet aggregation, hemoglobin interference, and other interferences on the test results.

[0016] Furthermore, by adding 5% trehalose to the photosensitive microsphere diluent, the luminescent microsphere diluent, and the biotin diluent, the first, second, and third components can be prepared as lyophilized reagents. Experimental data show that, compared to the liquid reagent, the stability is significantly improved after preparation as a lyophilized reagent.

[0017] In some embodiments of the present invention, the preparation process of the above-mentioned lyophilized reagent includes the following steps:

[0018] Cooling control: -40℃ for 50 minutes;

[0019] Pre-evacuate to 0.2 mbar;

[0020] Single drying: -25℃ / 360min, -20℃ / 420min, vacuum set at 0.1mbar;

[0021] Desorption drying: 35℃ for 300 min, with a vacuum of 0.3 mbar.

[0022] Furthermore, the homogeneous detection kit of the present invention also includes a separation reagent containing magnetic beads coated with anti-human erythrocyte (RBC) antibodies. The anti-human RBC antibodies are composed of an anti-human RBC monoclonal antibody targeting GPA and an anti-human RBC monoclonal antibody targeting H antigen, and the magnetic beads have a particle size of 0.1-0.5 μm. In a preferred embodiment of the present invention, the ratio of the anti-human RBC monoclonal antibody targeting GPA to the anti-human RBC monoclonal antibody targeting H antigen is 1:1, and the magnetic beads have a particle size of 0.3 μm. The magnetic beads are activated by EDC and NHS and then conjugated with the anti-human RBC antibodies. This separation reagent can rapidly pretreat arterial whole blood, resulting in clear plasma without hemolysis; and the resulting plasma is compatible with the above three components, exhibiting excellent detection performance.

[0023] Secondly, based on the above-mentioned homogeneous detection kit, the present invention provides a method for non-diagnostic immunoassay of target substances using arterial blood, comprising the following steps:

[0024] Arterial whole blood was mixed with magnetic beads coated with anti-human RBC antibody and then placed in a magnetic field for 1-2 minutes to separate and obtain clear plasma.

[0025] The obtained clarified plasma was mixed with the first, second and third components and then placed on a homogeneous chemiluminescence immunoassay analyzer for detection.

[0026] The detection principle of the above method is as follows: When the arterial blood sample being tested contains the antigen to be tested, a bridge is formed between the first component, the second component, and the third component: luminescent microsphere-detection antibody 1-antigen-biotin-labeled antibody 2-streptavidin-photosensitive microsphere. Under laser irradiation, energy is transferred from the photosensitive microsphere to the luminescent microsphere. After receiving energy, the surface of the luminescent microsphere undergoes an oxidation-reduction reaction, which in turn generates a light signal. The signal intensity is proportional to the content of the antigen to be tested in the sample. The light signal is captured by the photosensitive element, and the concentration of the analyte in the sample is calculated by mathematical fitting.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] The homogeneous chemiluminescence detection kit of this invention can be directly used to detect arterial blood after it has been sorted by the specific magnetic beads of this invention, and it is not affected by other substances in the arterial blood sample, thus ensuring the accuracy of the test results and providing strong support for clinical diagnosis.

[0029] This invention employs a special lyophilization process to produce high-quality lyophilized reagents from the aforementioned kit. The detection performance remains unaffected by the lyophilization process itself, ensuring the reagent's high efficiency and reliability. The lyophilized reagents exhibit rapid reconstitution and excellent reconstitution stability, with detection results showing extremely high consistency with the liquid reagent before lyophilization. Furthermore, these lyophilized reagents possess excellent stability, remaining stable for one year at room temperature (25°C) without any performance degradation. Even at temperatures as high as 45°C, they can be stably stored for over 40 days. This characteristic makes them ideal for transportation, storage, and use in high-temperature environments, significantly reducing transportation and storage costs and improving ease of use. Attached Figure Description

[0030] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0031] Figure 1 The results of the correlation comparison between the cTnI lyophilized homogeneous kit provided in Example 5 and the magnetic bead-treated arterial blood plasma and homologous serum were obtained.

[0032] Figure 2 The results of the correlation between the cTnI lyophilized homogeneous kit provided in Example 5 and the cTnI kit from the internationally renowned manufacturer Beckman Coulter, used to test the correlation between the results of the test on the magnetic bead-treated arterial blood plasma and the results of the test on the homologous venous serum.

[0033] Figure 3 The results of the correlation comparison between arterial blood plasma treated with magnetic beads and homologous serum were obtained using the myoglobin lyophilized homogeneous kit provided in Example 6.

[0034] Figure 4 The results show the correlation between the myoglobin lyophilized homogeneous kit provided in Example 6 and the myoglobin kit from Roche, a well-known international manufacturer, in testing arterial blood plasma treated with magnetic beads and homologous venous serum. Detailed Implementation

[0035] The technical solution of the present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs; the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the invention. The terms “comprising” and “having”, and any variations thereof, are intended to cover non-exclusive inclusion.

[0037] In this invention, technical terms such as "first" and "second" are used only to distinguish different objects and should not be construed as indicating or implying relative importance or implicitly specifying the number, specific order, or primary and secondary relationship of the indicated technical features.

[0038] To address the problem that existing immunoassay techniques, such as chemiluminescence immunoassay, cannot utilize arterial blood for detection, this invention develops a method and kit for immunoassay of target substances using arterial blood. Through improvements in arterial blood pretreatment and detection reagents, the detection of target substances in arterial blood has been successfully achieved, and the detection results are accurate and reliable.

[0039] The homogeneous detection kit provided in this embodiment of the invention includes the following components:

[0040] The separation reagent contains magnetic beads coated with anti-human RBC antibodies. The anti-human RBC antibodies are composed of anti-human RBC monoclonal antibodies targeting GPA and anti-human RBC monoclonal antibodies targeting H antigen. The magnetic beads have a particle size of 0.1-0.5 μm and are coupled to the anti-human RBC antibodies after activation by EDC and NHS.

[0041] The first component is obtained by diluting luminescent microspheres bound to detection antibody 1 with a luminescent microsphere diluent, and the luminescent microsphere diluent contains 20mM-50mM 2-morpholinoethanesulfonic acid, 0.01%-0.2% KCl and 0.1%-2% NaHCO3; the luminescent microspheres bound to detection antibody 1 refer to those on the surface of the luminescent microspheres coated with detection antibody 1.

[0042] The second component is obtained by diluting the biotin-labeled detection antibody 2 with a biotin diluent, which contains 0.2mM-5mM dithiothreitol, 1mM-10mM CaCl2, 1mM-10mM adenine nucleoside, and 20μg / ml-100μg / ml mouse anti-human hemoglobin antibody.

[0043] The third component is obtained by diluting photosensitive microspheres containing streptavidin with a photosensitive microsphere diluent. Photosensitive microspheres containing streptavidin refer to photosensitive microspheres with streptavidin coated on their surface.

[0044] Preferably, the separation reagent, the first component, the second component, and the third component are all prepared into freeze-dried microspheres for preservation, and the diameter of the freeze-dried microspheres is 2 mm-4 mm.

[0045] This invention also provides a kit for immunoassay of target substances using arterial blood. Specifically, whole arterial blood is mixed with magnetic beads coated with anti-human erythrocyte antibodies and then separated in a magnetic field for 1-2 minutes to obtain clear plasma. The obtained clear plasma is then mixed with the first, second, and third components and placed on a homogeneous chemiluminescence immunoassay analyzer for detection.

[0046] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0047] Example 1

[0048] This example provides a homogeneous detection kit for immunoassay of a target substance using arterial blood, comprising at least the following:

[0049] 1. Separation reagent.

[0050] The separation reagent contains magnetic beads coated with anti-human RBC antibodies, wherein the anti-human RBC antibodies are composed of anti-human RBC monoclonal antibodies targeting GPA and anti-human RBC monoclonal antibodies targeting H antigen, and the particle size of the magnetic beads is 0.3 μm.

[0051] The above separation reagent is prepared as follows:

[0052] (1) Cleaning: Take 500 μL of magnetic beads with a particle size of 0.3 μm, remove the original storage solution by strong magnetic adsorption, add 1.5 mL of activation buffer (10 mM MES buffer, pH=6.0), mix by sonication (20 kHz, 10 s), remove the supernatant by strong magnetic adsorption, and repeat this process twice.

[0053] (2) Activation: Add 1 mL of activation buffer to the cleaned magnetic bead precipitate, sonicate (20 kHz, 2 min) to mix, add 23 μL of EDC (5 mg / mL), vortex to mix, place on a shaker at 100 rpm and 37 ℃ for 5 min, add 46 μL of NHS (5 mg / mL), vortex to mix, place on a shaker at 100 rpm and 37 ℃ for 25 min.

[0054] (3) Coupling: After activation, the activation solution is discarded by strong magnetic adsorption. The solution is washed twice with pure water according to step (1). 1 mL of coupling buffer (20 mM Hepes buffer, pH=7.0) is added to the magnetic bead precipitate. After sonication (20 kHz, 2 min), 25 μg of anti-human RBC antibody (antibody: magnetic beads = 1:50, the antibody is composed of anti-human erythrocyte monoclonal antibody targeting GPA and anti-human erythrocyte monoclonal antibody targeting H antigen with a mass ratio of 1:1) is added. The mixture is vortexed and placed on a shaker at 100 rpm and 37 ℃ for 3 h for coupling.

[0055] (4) Blocking: After coupling, add 200 μL of blocking solution (2% BSA) to the reaction system and continue to react on a shaker at 100 rpm and 37 °C for 30 min.

[0056] (5) Cleaning and storage: After sealing, the supernatant was discarded by strong magnetic adsorption. The magnetic beads were washed twice with 50mM glycine solution (pH=7.4) according to step (1). The storage solution (20mM Hepes + 0.5% BSA + 0.1% Triton X-100) was added to the magnetic bead precipitate to make the final concentration of magnetic beads 5mg / mL. After sonication (20kHz, 2min), the mixture was stored at 2-8℃.

[0057] (6) Freeze-drying: Freeze-dried anti-human RBC immunomagnetic beads were obtained by freezing and vacuum drying with liquid nitrogen.

[0058] 2. First component.

[0059] The first component contains luminescent microspheres bound to detection antibody 1 and a luminescent microsphere diluent.

[0060] The above-mentioned method for preparing luminescent microspheres incorporating detection antibody 1 includes the following steps:

[0061] (1) Microsphere cleaning: Take 1 mg of luminescent microspheres and suspend them in 20 mM MES buffer. After sonication for 10 s, centrifuge to precipitate the microspheres and remove the supernatant (20000×g, 15 min). Repeat the above sonication and centrifugation precipitation operation 2-3 times to thoroughly remove the storage buffer.

[0062] (2) Microsphere activation: The luminescent microspheres were resuspended in 20 mM MES buffer, 10 μl of 10 mg / ml EDC solution was added, and then 100 μl of 10 mg / ml NHS solution was added. The microspheres were shaken and mixed, and incubated at 25°C in the dark at 40 r / min for 30 minutes. After the reaction was completed, unreacted EDC and NHS were removed by washing in step (1).

[0063] (3) Antibody coating: Take 100 μg of detection antibody 1 and put it into the activated luminescent microspheres. Shake to mix the microspheres and incubate at 40 r / min for 2 h in the dark at 25 °C. After the reaction is completed, remove the unreacted antibody by washing in step (1).

[0064] (4) Blocking: After the coating reaction is completed, a blocking solution containing 5% BSA + 5% glycine is added, and the mixture is incubated at 40 r / min for 2 h at 25 °C in the dark to block the unreacted active sites on the microspheres.

[0065] (5) Cleaning and storage: The blocked luminescent microspheres were cleaned with 10mM PBS (containing 1% Tween 20 and 1% BSA) according to the cleaning method in step (1) to remove unreacted antibodies and blocking solution; after cleaning, the microspheres were prepared to 1mg / ml and stored at 4℃ for later use.

[0066] The preparation method of the above-mentioned luminescent microsphere diluent is as follows: (1) Take a 1L beaker and add 800mL of ultrapure water to it; (2) Weigh 9.762g of 2-morpholinoethanesulfonic acid and add it to the above beaker, stir and mix until completely dissolved, and adjust the pH to 6.0±0.1; (3) Weigh 1g of KCl and add it to the above beaker, stir and mix until completely dissolved; (4) Weigh 10g of NaHCO3 and add it to the above beaker, stir and mix until completely dissolved; (5) Weigh 10g of BSA and add it to the above beaker, stir and mix until completely dissolved; (6) Weigh 2g of TritonX-405 and add it to the above beaker, stir and mix until completely dissolved; (7) Take 1ml of ProClin 300 and add it to the above beaker, stir and mix until completely dissolved; (8) Adjust the pH to 6.0±0.1, make up to 1L, and filter to obtain the luminescent microsphere diluent.

[0067] The prepared luminescent microspheres bound to detection antibody 1 were diluted to 25 μg / ml with luminescent microsphere diluent to obtain the first component.

[0068] 3. Second component.

[0069] The second component contains biotin-labeled detection antibody 2 and biotin diluent.

[0070] The preparation of the biotin-labeled detection antibody 2 described above includes the following steps:

[0071] Take 500 μg of detection antibody 2, desalt it using a desalting column and replace it with labeling buffer (50 mM PBS, pH=7.4), add 2 μl of 10 mg / ml biotin-NHS, vortex to mix, and incubate at 25 °C at 40 r / min for 2 h. After the reaction is complete, remove unreacted biotin-NHS using a desalting column and store at 4 °C for later use.

[0072] The preparation method of the above biotin dilution solution is as follows: (1) Take a 1L beaker and add 800mL of ultrapure water to it; (2) Weigh 4.766g of 4-hydroxyethylpiperazine ethanesulfonic acid and add it to the above beaker, stir and mix until completely dissolved, and adjust the pH to 7.0±0.1; (3) Weigh 9g of NaCl and add it to the above beaker, stir and mix until completely dissolved; (4) Weigh 154.2mg of dithiothreitol and add it to the above beaker, stir and mix until completely dissolved; (5) Weigh 222mg of CaCl2 and add it to the above beaker, stir and mix until completely dissolved; (6) Weigh 10g of BSA and add it to the above beaker, stir and mix until completely dissolved; (7) Weigh 2534.4mg of adenine nucleoside and add it to the above beaker, stir and mix until completely dissolved; (8) Add 50mg of mouse anti-human hemoglobin antibody to the above beaker, stir and mix until completely dissolved; (9) Add 1ml of ProClin Add 300 to the above beaker and stir until completely dissolved; (10) Adjust the pH to 7.0±0.1, make up to 1L, and filter to obtain the biotin dilution solution.

[0073] The prepared biotin-labeled detection antibody 2 was diluted to 1 μg / ml using biotin dilution buffer, which became the second component.

[0074] 4. The third component.

[0075] The third component contains photosensitive microspheres bound with streptomycin and a photosensitive microsphere diluent.

[0076] The preparation of the above-mentioned photosensitive microspheres containing streptomycin includes the following steps:

[0077] (1) Microsphere cleaning: Take 1 mg of photosensitive microspheres and suspend them in 20 mM MES buffer. After sonication for 10 s, centrifuge to precipitate the microspheres and remove the supernatant (20000×g, 15 min). Repeat the above sonication and centrifugation precipitation operation 2-3 times to thoroughly remove the storage buffer.

[0078] (2) Microsphere activation: Resuspend the photosensitive microspheres in 20mM MES buffer, add 10μl of 10mg / ml EDC solution, then add 100μl of 10mg / ml NHS solution, shake to mix the microspheres, and incubate at 25℃ in the dark at 40r / min for 30 minutes. After the reaction is complete, remove unreacted EDC and NHS by washing in step (1).

[0079] (3) Streptavidin coating: 100 μg of streptavidin was added to the activated photosensitive microspheres, the microspheres were shaken and mixed, and incubated at 40 r / min for 2 h in the dark at 25 °C. After the reaction was completed, the unreacted streptavidin was removed by the washing method in step (1).

[0080] (4) Blocking: After the coating reaction is completed, a blocking solution containing 5% BSA + 5% glycine is added, and the mixture is incubated at 40 r / min for 2 h at 25 °C in the dark to block the unreacted active sites on the microspheres.

[0081] (5) Cleaning and storage: The blocked photosensitive microspheres were cleaned with 10mM PBS (containing 1% Tween 20 and 1% BSA) according to the cleaning method in step (1) to remove unreacted streptavidin and blocking solution; after cleaning, the microspheres were prepared to 1mg / ml and stored at 4℃ for later use.

[0082] The preparation method of the above photosensitive microsphere diluent is as follows: (1) Take a 1L beaker and add 800mL of ultrapure water to it; (2) Weigh 4.766g of 4-hydroxyethylpiperazine ethanesulfonic acid and add it to the above beaker, stir and mix until completely dissolved, and adjust the pH to 7.0±0.1; (3) Weigh 9g of NaCl and add it to the above beaker, stir and mix until completely dissolved; (4) Weigh 10g of BSA and add it to the above beaker, stir and mix until completely dissolved; (5) Weigh 2g of TritonX-405 and add it to the above beaker, stir and mix until completely dissolved; (6) Take 1ml of ProClin 300 and add it to the above beaker, stir and mix until completely dissolved; (7) Weigh 5ml of bovine serum and add it to the above beaker, stir and mix until completely dissolved; (8) Adjust the pH to 7.0±0.1, make up to 1L, and filter to obtain the photosensitive microsphere diluent.

[0083] The photosensitive microspheres containing streptomycin prepared above were diluted to 50 μg / ml using a photosensitive microsphere diluent, which became the third component.

[0084] Example 2

[0085] Based on Example 1, this example provides a lyophilized homogeneous chemiluminescence detection kit. Specifically, compared to Example 1, the first, second, and third components are all prepared as lyophilized reagents. The specific preparation method is as follows:

[0086] Trehalose was added to the luminescent microsphere diluent of Example 1 to a final concentration of 5% to obtain a luminescent microsphere lyophilization buffer; the luminescent microspheres bound to detection antibody 1 were diluted to 25 μg / ml with the luminescent microsphere lyophilization buffer to obtain the first component.

[0087] Trehalose was added to the biotin dilution buffer in Example 1 to achieve a final concentration of 5%, thereby obtaining biotin lyophilization buffer; the biotin-labeled detection antibody 2 was diluted to 1 μg / ml with the biotin lyophilization buffer, which is the second component.

[0088] Trehalose was added to the photosensitive microsphere diluent of Example 1 to a final concentration of 5% to obtain a photosensitive microsphere lyophilization buffer; the photosensitive microspheres bound with streptomycin were diluted with the photosensitive microsphere lyophilization buffer to a concentration of 50 μg / ml, which is the third component.

[0089] Then, the first, second, and third components were prepared into a lyophilized reagent using the following lyophilization process:

[0090] Cooling control: -40℃ / 50min;

[0091] Pre-vacuum: 0.2 mbar;

[0092] Single drying: -25℃ / 360min, -20℃ / 420min, vacuum set at 0.1mbar;

[0093] Desorption drying: 35℃ for 300 min, with a vacuum of 0.3 mbar.

[0094] The first, second, and third components were all lyophilized in 50 μl increments. After lyophilization, the lyophilized microspheres were dispensed using a dedicated reagent dispenser, with each dispenser containing three lyophilized microspheres prepared from the first, second, and third components, respectively. The dispensers were then sealed with aluminum foil and stored at room temperature away from light for later use.

[0095] Example 3

[0096] Based on the kit in Example 1, this example provides a homogeneous chemiluminescence detection method for immunoassay of target substances using arterial blood, comprising the following steps:

[0097] Add the corresponding volume of pure water to the reaction tube containing the lyophilized magnetic beads to reconstitute them, and shake to mix until the magnetic beads are uniformly dispersed. Draw arterial whole blood from the anticoagulated blood collection tube into the uniformly dispersed magnetic bead suspension, with a mass-to-volume ratio of magnetic beads to whole blood of 2.0 mg: 500 μL. Shake to mix, and then place in a magnetic field for separation for 1-2 minutes to obtain clear plasma.

[0098] Clarified plasma was collected and mixed with the first, second, and third components, and then detected on a homogeneous chemiluminescence immunoassay analyzer. Qualitative and quantitative analysis of the target substances was performed based on the luminescence.

[0099] Example 4

[0100] Referring to Example 1, this example provides a liquid homogeneous detection kit with cTnI as the target substance, that is, both detection antibody 1 and detection antibody 2 are anti-cTnI antibodies.

[0101] In this embodiment, both detection antibody 1 and detection antibody 2 were purchased from Phytobio, with detection antibody 1 having catalog number F43 and detection antibody 2 having catalog number F33.

[0102] Example 5

[0103] Referring to Examples 1 and 2, this example provides a lyophilized homogeneous detection kit with cTnI as the target substance, wherein detection antibody 1 and detection antibody 2 are both anti-cTnI antibodies, and the antibodies used are the same as those in Example 4.

[0104] Example 6

[0105] Referring to Examples 1 and 2, this example provides a lyophilized homogeneous detection kit with myoglobin as the target substance, wherein both detection antibody 1 and detection antibody 2 are anti-myoglobin antibodies.

[0106] In this embodiment, both detection antibody 1 and detection antibody 2 were purchased from HaiTai Biotechnology. The product number of detection antibody 1 was 4E2cc and the product number of detection antibody 2 was 7C3cc.

[0107] Based on the method in Example 3, the performance of the lyophilized homogeneous chemiluminescence immunoassay kits in Examples 5 and 6 was tested. The homogeneous chemiluminescence immunoassay analyzer used in the following tests was purchased from Suzhou Weidu Biotechnology Co., Ltd.; the serum of the venous blood used was prepared by collecting blood from the vein using a vacuum blood collection tube without a coagulant, placing it in a 37°C water bath for half an hour to promote full coagulation, and then centrifuging it in a centrifuge at 3000 rpm / 10 min.

[0108] (1) Detection of lyophilized cTnI kit.

[0109] Tear open the aluminum foil seal of the lyophilized reagent, add 150 μl of purified water to reconstitute the lyophilized microspheres; then add clarified plasma obtained by treating arterial blood with magnetic beads and serum of homologous venous blood respectively, and perform the measurements on a homogeneous chemiluminescence immunoassay analyzer.

[0110] The results are shown in Table 1 and Figure 1 As shown, the measurement deviation of the vast majority of samples is within ±10%, and the measurement deviation of a very few low-value samples is within ±10% to ±15%. The correlation coefficient R0 2 The value was 0.9977, indicating high consistency. There was no significant difference between the two measurements, demonstrating that the lyophilized cTnI kit provided by this invention can achieve arterial blood detection.

[0111] Table 1. Comparison of results from arterial blood plasma and homologous serum tested using the lyophilized cTnI kit.

[0112]

[0113] Note: The unit of measurement is ng / ml.

[0114] Serum from homologous venous blood was collected and tested using a Beckman Coulter high-sensitivity cTnI assay kit on an Access 2 automated immunoassay analyzer. The results are shown in Table 2 and... Figure 2 As shown, the deviations between the arterial blood test results of the kit of the present invention and the venous serum test results of the Beckman cTnI kit are within ±15%, and the correlation coefficient R is [missing value]. 2 The value was 0.9922, indicating high consistency. There was no significant difference between the two measurements, demonstrating that the lyophilized cTnI kit provided by this invention is accurate and reliable for arterial blood detection.

[0115] Table 2. Comparison of results from arterial blood plasma tested with the lyophilized cTnI kit and homologous serum tested with the Beckman Coulter cTnI kit.

[0116]

[0117] Note: The unit of measurement is ng / ml.

[0118] (2) Detection of lyophilized myoglobin using a kit.

[0119] Tear open the aluminum foil seal of the lyophilized reagent, add 150 μl of purified water to reconstitute the lyophilized microspheres; then add clarified plasma obtained by treating arterial blood with magnetic beads and serum of homologous venous blood respectively, and perform the measurements on a homogeneous chemiluminescence immunoassay analyzer.

[0120] The results are shown in Table 3 and Figure 3 As shown, the measurement deviation of most samples is within ±10%, and the correlation coefficient R0 is... 2 The value was 0.9959, indicating high consistency, which shows that the lyophilized myoglobin homogeneous kit provided by this invention can be used for the detection of arterial blood.

[0121] Table 3. Comparison of results from arterial blood plasma and homologous serum tested using the lyophilized myoglobin kit.

[0122]

[0123] Note: The unit of measurement is ng / ml.

[0124] Serum samples from homologous venous blood were collected and tested using the Roche myoglobin assay kit on a fully automated immunoassay analyzer, Cobas e 411. The results are shown in Table 4 and... Figure 4 As shown, the deviations between the arterial blood test results of the kit of the present invention and the venous serum test results of the Roche myoglobin kit are all within ±15%, and the correlation coefficient R is [missing value]. 2The value was 0.9927, indicating high consistency. There was no significant difference between the two measurements, demonstrating that the lyophilized myoglobin kit provided by this invention is accurate and reliable for arterial blood testing.

[0125] Table 4. Comparison of results between arterial blood plasma tested with the lyophilized myoglobin kit and homologous serum tested with the Roche myoglobin kit.

[0126]

[0127] Note: The unit of measurement is ng / ml.

[0128] (3) Repeatability test of the kit.

[0129] The aluminum foil seal of the lyophilized reagent in Example 4 was torn open, and 150 μl of purified water was added to reconstitute the lyophilized microspheres. Low, medium, and high-value arterial blood samples treated with magnetic beads were then added, and measurements were performed on a homogeneous chemiluminescence immunoassay analyzer. The results are shown in Table 5. The coefficients of variation for low, medium, and high-value samples tested using the lyophilized cTnI homogeneous detection kit were all <3%, indicating excellent repeatability.

[0130] Table 5. Repeatability test results of the lyophilized troponin I kit

[0131]

[0132] (4) Stability test of the kit.

[0133] The liquid reagent from Example 4 and the lyophilized reagent from Example 5 were dispensed into sealed containers, and the openings were sealed with sealing film to further isolate them from air. The containers were placed in a 45°C constant temperature incubator for thermally accelerated stability testing. The results were measured after 40 days, and the test results were compared and analyzed with the test results before thermal acceleration at 45°C.

[0134] The results are shown in Table 6. After 40 days of accelerated heating at 45°C, the signal values ​​of the tested antigen samples and whole blood samples decreased by less than ±10%, while the signal value of the liquid reagent decreased by nearly 90%, indicating that the stability of the homogeneous lyophilized reagent is significantly better than that of the liquid reagent.

[0135] Table 6. Results of thermally accelerated stability of lyophilized and liquid troponin I reagents

[0136]

[0137] In summary, the reagent kit provided by this invention can detect target substances in arterial blood, while exhibiting excellent functional sensitivity and detection range. It is unaffected by interference from other substances in arterial blood, ensuring the accuracy of test results. Furthermore, the reagent is in lyophilized solid form, allowing for room temperature transportation and storage. Therefore, the solution of this invention has excellent application prospects in clinical blood testing.

[0138] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. A homogeneous detection kit for immunoassay of a target substance using arterial blood, characterized in that, At least the following reagents are included: The first component comprises luminescent microspheres bound to detection antibody 1 and a luminescent microsphere diluent, wherein the luminescent microsphere diluent contains 2-morpholine ethanesulfonic acid, KCl and NaHCO3; The second component comprises a biotin-labeled detection antibody 2 and a biotin diluent containing dithiothreitol, CaCl2, adenine nucleoside, and mouse anti-human hemoglobin antibody; A third component containing photosensitive microspheres incorporating streptomycin; A separation reagent containing magnetic beads coated with anti-human erythrocyte antibodies, wherein the anti-human erythrocyte antibodies are composed of anti-human erythrocyte monoclonal antibodies targeting GPA and anti-human erythrocyte monoclonal antibodies targeting H antigen, and the particle size of the magnetic beads is 0.1-0.5 μm.

2. The homogeneous detection kit according to claim 1, characterized in that, In the first component, the concentration of the luminescent microspheres bound to detection antibody 1 is 10 μg / ml-100 μg / ml; in the second component, the concentration of the biotin-labeled detection antibody 2 is 0.1 μg / ml-10 μg / ml; and in the third component, the concentration of the photosensitive microspheres bound to streptomycin is 20 μg / ml-200 μg / ml.

3. The homogeneous detection kit according to claim 2, characterized in that, In the first component, the concentration of the luminescent microspheres bound to detection antibody 1 is 10 μg / ml-50 μg / ml; in the second component, the concentration of the biotin-labeled detection antibody 2 is 0.5 μg / ml-5 μg / ml; and in the third component, the concentration of the photosensitive microspheres bound to streptomycin is 20 μg / ml-100 μg / ml.

4. The homogeneous detection kit according to claim 3, characterized in that, In the first component, the concentration of the luminescent microspheres bound to detection antibody 1 is 20 μg / ml-40 μg / ml; in the second component, the concentration of the biotin-labeled detection antibody 2 is 1 μg / ml-3 μg / ml; and in the third component, the concentration of the photosensitive microspheres bound to streptomycin is 20 μg / ml-80 μg / ml.

5. The homogeneous detection kit according to claim 3, characterized in that, The luminescent microsphere diluent contains 20mM-50mM 2-morpholinoethanesulfonic acid, 0.01%-0.2% KCl, and 0.1%-2% NaHCO3. The biotin diluent contains 0.2mM-5mM dithiothreitol, 1mM-10mM CaCl2, 1mM-10mM adenine nucleoside, and 20μg / ml-100μg / ml mouse anti-human hemoglobin antibody.

6. The homogeneous detection kit according to claim 1, characterized in that, The third component contains a photosensitive microsphere diluent, and the photosensitive microsphere diluent, the luminescent microsphere diluent, and the biotin diluent all contain 5% trehalose. Furthermore, the first, second, and third components are all lyophilized reagents.

7. The homogeneous detection kit according to claim 6, characterized in that, The preparation process of the lyophilized reagent includes the following steps: Cooling control: -40℃ for 50 minutes; Pre-evacuate to 0.2 mbar; Single drying: -25℃ / 360min, -20℃ / 420min, vacuum set at 0.1mbar; Desorption drying: 35℃ for 300 min, with a vacuum of 0.3 mbar.

8. A method for non-diagnostic immunoassay of a target substance using arterial blood, characterized in that, The detection was performed using the homogeneous detection kit according to any one of claims 1-7.

9. The method according to claim 8, characterized in that, Includes the following steps: Arterial whole blood was mixed with magnetic beads coated with anti-human erythrocyte antibodies and then placed in a magnetic field for 1-2 minutes to separate, resulting in clear plasma. The clarified plasma was mixed with the first, second and third components and then placed on a homogeneous chemiluminescence immunoassay analyzer for detection.

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