Heparin binding protein determination kit for peripheral blood

Abstract

The invention discloses a heparin binding protein determination kit for peripheral blood as well as a preparation method and application of the heparin binding protein determination kit. The kit comprises an acridinium ester labeled heparin binding protein antibody, a magnetic particle labeled heparin binding protein (HBP) antibody, a pre-excitation liquid, an excitation liquid, an HBP series calibration product and an HBP series quality control product. The kit and the method are established by using a magnetic particle and acridinium ester chemiluminescence immunoassay technology based on the principle of a double-antibody sandwich method. The method shows the analysis potential of peripheral blood in on-site and bedside fields, and is of great significance to pre-hospital first aid and early screening of sepsis patients.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a heparin binding protein assay kit for peripheral blood, a preparation method thereof and an application thereof. Background Art

[0002] Sepsis and septic shock remain one of the major public health problems facing critical care medicine. Early diagnosis and treatment are the key to improving the prognosis of patients with sepsis and septic shock. In order to diagnose and treat patients with sepsis or those who may develop sepsis more quickly and accurately, the American Society of Critical Care Medicine and the European Society of Intensive Care Medicine have released new diagnostic criteria for sepsis and septic shock. The sequential organ failure assessment (SOFA) and acute physiology and chronic health evaluation scoring system (APACHE II) for sepsis diagnosis are the most commonly used organ failure scores in ICUs. However, the existing microbial culture and traditional infection indicators cannot meet the clinical needs for timely, accurate and rapid diagnosis due to time delays, low sensitivity and lack of specificity. Early and accurate diagnosis of sepsis and septic shock remains challenging. The study by Linder et al. (Adam Linder et al, Critical Care Medicine, 2015, 43: 2378-2386) showed that heparin-binding protein, which is involved in inflammatory response, can effectively predict the occurrence of sepsis and septic shock, and is a good clinical diagnostic indicator.

[0003] Enzyme-linked immunosorbent assay can accurately detect the concentration of heparin-binding protein in samples, but in actual operation, the ELISA experiment takes a long time, generally requiring 4 to 5 hours to obtain the test results. As an important indicator for predicting sepsis and inflammatory response, it cannot meet the needs of rapid testing in outpatient or emergency departments. Although the immunofluorescence chromatography method can be used for rapid testing, due to the use of a heterogeneous reaction system, the precision of the test is poor, and the interference cannot be removed from the operating procedure, so the accuracy of the test cannot be guaranteed.

[0004] Heparin-Binding Protein (HBP), also known as azurocidin or CAP37, originates from neutrophils and is mainly stored in azurocidin granules, with a small portion stored in secretory vesicles. HBP is a member of the serine protease family and has no protease activity. It has a high affinity for lipopolysaccharide lipid A. Neutrophils degranulate after activation, releasing HBP. When acute bacterial infection occurs, the concentration of HBP in the blood can increase significantly within 1-2 hours, while HBP does not increase or increases slightly during viral infection. HBP also acts as a pathogenic factor. When the patient receives effective treatment, the HBP concentration drops rapidly. Therefore, detecting the HBP content in the patient's blood can assist in the diagnosis and prediction of acute bacterial infection, the judgment of the severity of infection, and the monitoring of the efficacy of antibiotics.

[0005] For patients who monitor their HBP levels, venous blood sampling is not suitable for repeated use. Peripheral blood sampling is convenient and causes less wounds to people, making it a better source of blood samples. However, there are significant differences in the composition of peripheral blood and venous blood. For example, in general peripheral blood, red blood cells, hemoglobin, hematocrit, mean corpuscular hemoglobin content, mean corpuscular hemoglobin concentration, reticulocyte count and neutrophil content are higher than those in venous blood. Platelets, mean corpuscular volume, lymphocyte content, eosinophil content and basophil content are lower than those in venous blood. Therefore, although there are some HBP detection products that can measure the HBP content in venous serum or plasma, these products cannot accurately detect the HBP content in peripheral blood. Compared with serum, plasma and whole blood samples, the analytical potential of peripheral blood in the field / bedside field is of great significance for pre-hospital emergency treatment and early screening of patients with sepsis.

[0006] Chinese invention patent 202010795512.7 discloses a magnetic microparticle chemiluminescence detection kit for HBP and its preparation method. The blank limit, linear range, hook effect, specificity (bilirubin, triglyceride, total protein, rheumatoid factor) and correlation with imported ELISA kits of the product were studied, but the consistency, correlation, regression analysis, hypothesis testing, etc. of venous blood and peripheral blood were not studied. The study of peripheral blood is particularly important.

[0007] Therefore, it is particularly important to invent a product for rapid and accurate detection of peripheral blood; establishing a detection method for quantitatively determining the content of heparin binding protein (HBP) in peripheral blood can meet the needs of faster and more accurate prediction of whether a patient has sepsis and determine the severity of sepsis. Summary of the invention

[0008] The object of the present invention is to provide a peripheral blood heparin binding protein assay kit, a preparation method and application thereof, so as to solve the above-mentioned problems existing in the prior art.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] In one aspect, the present invention provides a heparin binding protein assay kit for peripheral blood, comprising an acridinium ester-labeled heparin binding protein (HBP) antibody, a magnetic particle-labeled HBP antibody, a pre-excitation solution, an excitation solution, an HBP series calibrator, and an HBP series quality control product.

[0011] In some embodiments, an acridinium ester-labeled HBP antibody is prepared as follows:

[0012] S1. Take 10 μL diluted acridinium ester (NSP-DMAE-NHS) stock solution (2.5 mg / ml), add 90 μL anhydrous DMSO (or DMF) and dilute 10 times to prepare acridinium ester working solution (0.25 mg / ml);

[0013] S2. Dilute 50 μg of HBP antibody to 300 μL using 0.2 M NaHCO3 (pH = 9.0) solution, and add 10 μL of acridinium ester working solution (0.25 mg / ml);

[0014] S3. Wrap in tin foil and mark at room temperature for 0.5 to 1h;

[0015] S4. Quench the reaction, add 100 μL of labeling termination buffer (10% lysine 0.2 M NaHCO3 (pH = 9.0) solution), and mix at room temperature for 30 minutes;

[0016] S5. Purification of the above acridinium ester-conjugated antibody sample

[0017] 1) Select a 250 ml beaker, inject 150 ml of 0.2 M pH 9.4 carbonate-bicarbonate buffer into it, select a dialysis cassette with a cutoff of 3.5 KDa and take it out of the protective bag, and stand the dialysis cassette at the bottom of the beaker so that the liquid submerges the dialysis cassette membrane;

[0018] 2) While the dialysis cassette is soaking, the syringe is used to absorb all the acridinium ester-antibody labeling system samples, and the syringe is inverted to expel excess air and pop out bubbles, and then placed aside for use;

[0019] 3) After the dialysis cassette is soaked, spread absorbent paper on the laboratory table, take out the dialysis cassette, and gently tap the cassette upright and upside down on the absorbent paper to remove excess buffer. The time should be controlled within 1 minute.

[0020] 4) Slowly insert the needle tip of the syringe into the sampling port on the side of the dialysis box, and the syringe will suck the air in the cavity. Invert the syringe to expel excess air and pop out bubbles;

[0021] 5) Slowly insert the needle tip of the syringe into the sample inlet on the side of the dialysis box, inject all the sample (200-500 μg), and then suck out as much air as possible without sucking out the sample;

[0022] 6) Select a 250 ml beaker, add 300-400 times the sample volume of 0.2 M pH 9.4 carbonate-bicarbonate buffer, place the dialysis cassette upright and fully immersed in it, place the small rotor of the magnetic stirrer against the wall of the beaker and put it into the bottom of the beaker, cover the beaker with light-proof paper, turn on the magnetic stirrer, adjust the speed to 150-300 rpm, place the beaker in the center of the magnetic stirrer, and dialyze at room temperature for 0.5 h;

[0023] 7) Take out the dialysis cassette, discard the buffer in the beaker, add 250-350 times of 0.2M pH 9.4 carbonate-bicarbonate buffer, and repeat step 6) for dialysis at room temperature for 1 hour;

[0024] 8) Take out the dialysis cassette, discard the buffer in the beaker, add 200-300 times 0.2M pH 9.4 carbonate-bicarbonate buffer, and repeat step 6) for dialysis at room temperature for 2 hours;

[0025] 9) Take out the dialysis cassette, discard the buffer in the beaker, add 400-600 times 0.2M pH 9.4 carbonate-bicarbonate buffer, and dialyze at 4°C for 12-16 hours;

[0026] 10) After dialysis is completed, discard the dialysate, take out the dialysis box, insert a new syringe into the sampling port on the other side of the dialysis box, inject appropriate air into it, put the needle in the appropriate position, turn the box to the appropriate position, turn it upside down, and take out the sample with the syringe;

[0027] 11) First, add 0.5 ml of 0.2 M pH 9.4 carbonate-bicarbonate buffer into the cavity, stir with a magnetic stirrer for 5 min, and then recover; then add 0.3 ml of 0.2 M pH 9.4 carbonate-bicarbonate buffer into the cavity, stir with a magnetic stirrer for 4 min, and then recover; finally, add 0.2 ml of 0.2 M pH 9.4 carbonate-bicarbonate buffer into the cavity, stir with a magnetic stirrer for 3 min, and then recover;

[0028] 12) Combine all the recovered solutions, mark them as acridinium ester labeled mother solution, and determine the protein concentration.

[0029] In some embodiments, magnetic particle-labeled HBP antibodies are prepared by an NHS activation step and a protein coupling step.

[0030] Preferably, NHS activation is performed as follows:

[0031] 1) After fully mixing the magnetic beads, take 100 μL of the magnetic bead suspension into a 1.5 mL centrifuge tube, place it on a magnetic rack, and remove the supernatant after solid-liquid separation;

[0032] 2) Wash the magnetic beads twice with 200 μL MES solution (50 mM, pH 5.0) and discard the supernatant;

[0033] 3) Add 50 μL of freshly prepared EDC solution and 50 μL of NHS solution to the centrifuge tube containing magnetic beads, vortex to mix to fully suspend the magnetic beads, and shake and mix at room temperature for 30 minutes;

[0034] 4) After the reaction is completed, add 100 μL of the above MES solution and wash twice;

[0035] Preferably, protein coupling is performed as follows:

[0036] 1) Place the centrifuge tube in a magnetic separator, remove the supernatant, add 100 μL of protein solution (50 mM MES, pH 5.0) and mix gently;

[0037] 2) Mix by shaking at room temperature for 2 h, or overnight at 4 °C;

[0038] 3) After the reaction, the supernatant was removed by magnetic separation, and 100 μL MES buffer (50 mM, pH 5.0) was added to wash the magnetic beads three times;

[0039] 4) After the reaction is completed, place the centrifuge tube on a magnetic separation rack, let it stand for 2 minutes, and discard the supernatant; at room temperature, add 100 μL of ethanolamine solution (50 mM, pH 8.0) and rotate and mix for 60 minutes or Tris buffer (50 mM, pH 7.4) and rotate and mix for 15 minutes. This step is used to block excess active sites;

[0040] 5) After the reaction, wash 4 times with 100 μL PBS or 50 mM Tris;

[0041] 6) Add appropriate amount of PBS or Tris preservation solution and store at 4°C;

[0042] 7) Dilute the antibody-magnetic particle conjugate with magnetic particle diluent (0.05M Tris, 1% sodium chloride, 2% BSA, 4% trehalose, 0.05% TritonX-100, 0.5% Proclin300) to make the antibody concentration 0.5 mg / mL and store in a 4°C refrigerator.

[0043] In some embodiments, the HBP series calibrators and quality control materials are prepared as follows: recombinant or natural heparin binding protein is dissolved in a diluent containing 50mM pH7.3 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 0.5% lauryl alcohol polyoxyethylene ether, 50mM potassium chloride, 2% sucrose, 4% arginine and 0.2% Proclin300, and prepared into calibrators with concentrations of 0, 5, 50, 100, 200, and 300 ng / mL, respectively, and quality control materials with concentrations of 8 ng / mL and 40 ng / mL, respectively.

[0044] In some embodiments, the pre-stimulation solution comprises 0.1 mol / L HNO3 and 0.1% H2O2, and the stimulation solution comprises 0.25 mol / L NaOH and 2% Triton X-100.

[0045] In some embodiments, the kit further comprises a diluent and a buffer.

[0046] In some embodiments, the diluent for magnetic particle labeling comprises 0.05M Tris, 1% sodium chloride, 2% BSA, 4% trehalose, 0.05% Triton X-100 and 0.5% Proclin300; the diluent for HBP series calibrators and quality controls comprises 50mM pH7.3 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 0.5% polyoxyethylene lauryl alcohol ether, 50mM potassium chloride, 2% sucrose, 4% arginine and 0.2% Proclin300.

[0047] In some embodiments, the buffer is selected from 0.2 M carbonate-bicarbonate buffer (pH 9.4), MES buffer (50 mM, pH 5.0) and Tris buffer (50 mM, pH 7.4).

[0048] In one aspect, the present invention provides use of the kit in preparing a product for detecting HBP in peripheral blood of a patient.

[0049] In some embodiments, the product includes the kit and instructions for use, etc.

[0050] In some embodiments, the patient has sepsis or septic shock.

[0051] In one aspect, the present invention provides a method for detecting heparin binding protein in peripheral blood of a patient based on magnetic microparticle acridinium ester chemiluminescence, the method comprising the following steps:

[0052] 1) Add 50 volumes of calibrator, quality control material or sample to be tested into the test tube;

[0053] 2) Add 50 volumes of magnetic particles coated with antibodies to the test tube;

[0054] 3) Add 100 volumes of acridinium ester-labeled antibody to the test tube;

[0055] 4) After mixing, incubate at 37°C for 20 minutes;

[0056] 5) Add 300 volumes of cleaning solution (PBS + 0.05% Tween 20) to the test tube and mix well;

[0057] 6) Magnetic separation and removal of supernatant;

[0058] 7) Repeat steps 5) and 6) at least twice;

[0059] 8) Add pre-excitation solution (100 μL 0.1 mol / L HNO3+0.1% H2O2) and excitation buffer (100 μL 0.25 mol / L NaOH+2% Triton X-100) to the detection tube, and the amount of pre-excitation solution and excitation buffer added is 100 parts by volume respectively;

[0060] 9) Detect the luminous intensity.

[0061] In some embodiments, the patient has sepsis or septic shock.

[0062] The present invention is based on the experimental principle of the double antibody sandwich method, and uses magnetic particles and acridinium ester chemiluminescent immunoassay technology to establish a kit and a detection method for quantitatively determining the content of heparin binding protein (HBP) in peripheral blood. Since acridinium ester chemiluminescent immunoassay technology is a very accurate quantitative detection, the detection accuracy is higher, while the accuracy of general immunological detection is significantly lower than the result of acridinium ester luminescent immunoassay technology, and the time is long and inconvenient. At the same time, the present invention demonstrates the analytical potential of peripheral blood in the field / bedside field, which is of great significance for pre-hospital emergency and early screening of sepsis patients.

[0063] The beneficial effects of the present invention include at least the following:

[0064] The present invention uses magnetic particle chemiluminescence immunoassay to quantitatively determine the content of heparin binding protein in human peripheral blood in vitro, and is a new type of immunoassay kit. Compared with the traditional ELISA method, the method of the present invention generally takes 2-3 hours and has errors caused by human operation, etc., while the method of the present invention can be quickly detected on a fully automatic chemiluminometer or a semi-automatic chemiluminometer, the reagent has good applicability, and the linear range of the product is wider and the sensitivity is higher. Most importantly, the method of the present invention can detect peripheral blood, which can fill the gap in the field of peripheral blood detection of chemiluminescence products on the current market, and provide more choices for clinical detection in pediatrics, outpatient clinics, emergency departments, and infectious departments of hospitals. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 Shows the absolute bias of the test system and the reference system.

[0066] Figure 2 Shows the relative bias of the test system and the reference system.

[0067] Figure 3 A scatter plot showing the correlation between the assessment system and the reference system. DETAILED DESCRIPTION

[0068] Example 1: Kit preparation

[0069] S1 Preparation of acridinium ester-labeled HBP antibody

[0070] 1. Take 10 μL diluted acridinium ester (NSP-DMAE-NHS) stock solution (2.5 mg / ml), add 90 μL anhydrous DMSO (or DMF) and dilute 10 times to prepare acridinium ester working solution (0.25 mg / ml).

[0071] 2. Dilute 50 μg of HBP antibody to 300 μL using 0.2 M NaHCO 3 (pH=9.0) solution, and add 10 μL of acridinium ester working solution (0.25 mg / ml).

[0072] 3. Wrap in tin foil and mark at room temperature for 0.5 to 1 hour.

[0073] 4. Quench the reaction, add 100 μL of labeling termination buffer (10% lysine 0.2 M NaHCO3 (pH=9.0) solution), and mix at room temperature for 30 minutes.

[0074] 5. Purification of the above acridinium ester-conjugated antibody samples

[0075] 1) Select a 250 ml beaker, inject 150 ml of 0.2 M pH 9.4 carbonate-bicarbonate buffer into it, select a 3.5 KDa cut-off dialysis cassette (Thermo Scientific Slide-A-Lyzer dialysis cassette) and take it out of the protective bag, and stand the dialysis cassette at the bottom of the beaker with your hand so that the liquid submerges the dialysis cassette membrane. Do not touch the dialysis membrane with your hands during the whole process;

[0076] 2) While the dialysis cassette is soaking, the syringe is used to absorb all the acridinium ester-antibody labeling system samples, and the syringe is inverted to expel excess air and pop out bubbles, and then placed aside for use;

[0077] 3) After the dialysis cassette is soaked, spread absorbent paper on the laboratory table, take out the dialysis cassette, and gently tap the cassette upright and upside down on the absorbent paper to remove excess buffer. The time should be controlled within 1 minute.

[0078] 4) Slowly insert the needle tip of the syringe into the sampling port on the side of the dialysis box, and the syringe will suck the air in the cavity. Invert the syringe to expel excess air and pop out bubbles;

[0079] 5) Slowly insert the needle tip of the syringe into the sample inlet on the side of the dialysis box, inject all the samples (200-500 micrograms), and then suck the air in the cavity with the syringe. As much air as possible is sucked out of the cavity under the premise of ensuring that the sample is not sucked out; 6) Select a 250ml beaker, add 300-400 times the volume of the sample 0.2M pH 9.4 carbonate-bicarbonate buffer, place the dialysis box upright and fully immersed in it, and put the small rotor of the magnetic stirrer against the wall of the beaker and put it into the bottom of the beaker. The beaker is treated with light-proof paper on all sides, and the magnetic stirrer is turned on. After adjusting to a low speed of 150-300rpm, the above beaker is placed in the center of the magnetic stirrer and dialyzed at room temperature for 0.5h;

[0080] 7) Take out the dialysis cassette, discard the buffer in the beaker, add 250-350 times of 0.2M pH 9.4 carbonate-bicarbonate buffer, and repeat step 6) for dialysis at room temperature for 1 hour;

[0081] 8) Take out the dialysis cassette, discard the buffer in the beaker, add 200-300 times 0.2M pH 9.4 carbonate-bicarbonate buffer, and repeat step 6) for dialysis at room temperature for 2 hours;

[0082] 9) Take out the dialysis cassette, discard the buffer in the beaker, add 400-600 times 0.2M pH 9.4 carbonate-bicarbonate buffer, and dialyze at 4°C for 12-16 hours;

[0083] 10) After dialysis is completed, discard the dialysate, take out the dialysis box, insert a new syringe into the sampling port on the other side of the dialysis box, inject appropriate air into it, put the needle in the appropriate position, turn the box to the appropriate position, turn it upside down, and take out the sample with the syringe;

[0084] 11) First, add 0.5 ml of 0.2 M pH 9.4 carbonate-bicarbonate buffer to the cavity, stir with a magnetic stirrer for 5 minutes, and then recover. Then, add 0.3 ml of 0.2 M pH 9.4 carbonate-bicarbonate buffer to the cavity, stir with a magnetic stirrer for 4 minutes, and then recover. Finally, add 0.2 ml of 0.2 M pH 9.4 carbonate-bicarbonate buffer to the cavity, stir with a magnetic stirrer for 3 minutes, and then recover. After adding liquid three times, use a syringe to expel the air in the cavity so that the recovered liquid is in full contact with the inner wall of the membrane.

[0085] 12) Combine all the recovered solutions, mark them as acridinium ester labeled mother solution, and determine the protein concentration.

[0086] S2 Preparation of magnetic microparticles HBP antibody

[0087] 1. NHS activation

[0088] 1) After fully mixing the magnetic beads, take 100 μL of the magnetic bead suspension into a 1.5 mL centrifuge tube, place it on a magnetic rack, and remove the supernatant after solid-liquid separation;

[0089] 2) Wash the magnetic beads twice with 200 μL MES solution (50 mM, pH 5.0) and discard the supernatant;

[0090] 3) Add 50 μL of freshly prepared EDC solution and 50 μL of NHS solution to the centrifuge tube containing magnetic beads, vortex to mix to fully suspend the magnetic beads, and shake and mix at room temperature for 30 minutes;

[0091] 4) After the reaction is completed, add 100 μL of the above MES solution and wash twice (the activated state should not be stored for a long time and coupling needs to be performed immediately).

[0092] 2. Protein Coupling

[0093] 1) Place the centrifuge tube in a magnetic separator, remove the supernatant, add 100 μL of protein solution (50 mM MES, pH 5.0) and mix gently (for the first experiment, the optimal concentration needs to be explored, and the recommended concentration is 1-10 mg / mL); 2) Shake and mix at room temperature for 2 hours, or shake and mix at 4°C overnight;

[0094] 3) After the reaction, the supernatant was removed by magnetic separation, and 100 μL MES buffer (50 mM, pH 5.0) was added to wash the magnetic beads three times;

[0095] 4) After the reaction is completed, place the centrifuge tube on a magnetic separation rack, let it stand for 2 minutes, and discard the supernatant. At room temperature, add 100 μL of ethanolamine solution (50 mM, pH 8.0) and rotate and mix for 60 minutes or Tris buffer (50 mM, pH 7.4) and rotate and mix for 15 minutes. This step is used to block excess active sites;

[0096] 5) After the reaction is completed, wash 4 times with 100 μL PBS or 50 mM Tris. Under the premise of not affecting downstream applications, 0.1-0.5% BSA can be added to the washing buffer as a blocking protein, or 0.1% Tween-20 or Triton-100 can be added to reduce nonspecific adsorption;

[0097] 6) Add an appropriate amount of PBS or Tris preservation solution (the amount of preservation solution added can be determined according to needs to adjust the concentration of coupled ligand magnetic beads) and store at 4°C. If the fixed biological ligand is stable, 0.02% (w / v) sodium azide can be added to the preservation solution as an antibacterial agent.

[0098] 7) Dilute the antibody-magnetic particle conjugate to 0.5 mg / mL (antibody concentration) with magnetic particle diluent (0.05 M Tris, 1% sodium chloride, 2% BSA, 4% trehalose, 0.05% Triton X-100, 0.5% Proclin 300) and store in a 4°C refrigerator.

[0099] S3 Preparation of pre-excitation solution

[0100] Pre-stimulation solution: 100μL 0.1mol / L HNO3+0.1%H2O2

[0101] S4 Preparation of excitation solution

[0102] Stimulation solution: 100 μL 0.25 mol / L NaOH + 2% Triton X-100

[0103] Preparation of S5 Calibrators and Quality Controls

[0104] The commercially available HBP antigen was prepared into a series of calibrators with calibrator diluent, with concentrations of 0 ng / mL, 5 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 300 ng / mL, respectively. The composition of the calibrator diluent was 50 mM pH 7.3 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 0.5% lauryl alcohol polyoxyethylene ether, 50 mM potassium chloride, 2% sucrose, 4% arginine, and 0.2% Proclin-300. The high-concentration HBP sample (80 ng / mL) was diluted to 8 ng / mL and 40 ng / mL with the calibrator diluent, which were used as low-value quality control and high-value quality control, respectively.

[0105] S6 Establishment of HBP standard curve

[0106] Take 50 μL of antibody-magnetic particles (20 μg / mL), add 50 μL of calibrant and 100 μL of paired antibody-acridinium ester luminescent agent, incubate the reaction at 37°C for 20 minutes, use a magnet to attract the magnetic particles and their magnetic particle conjugates to the bottom of the test tube, and use 300 μL of washing buffer (PBS+0.05% Tween 20) to gently wash the test tube to remove free substances. Then add 100 μL of pre-excitation solution and excitation solution respectively, and detect the luminescence value (RLU). The luminescence value is positively correlated with the analyte concentration in the sample, and a standard curve can be established based on the concentration and luminescence value.

[0107] Example 2: Product Clinical Evaluation

[0108] 1. Test products

[0109] The test reagent is a heparin binding protein (HBP) assay kit (magnetic microparticle chemiluminescence method) prepared according to Example 1. The reference reagent is a heparin binding protein assay kit (immunofluorescence dry quantitative method) of Zhonghan Shengtai Biotechnology Co., Ltd. (Zhejiang Medical Device No. 20182400180).

[0110] 2. Selection of trial participants

[0111] Homologous blood samples were collected from 90 patients aged 18 years and above who were admitted to the ICU of Linfen People's Hospital from May 1, 2023 to December 31, 2023, including peripheral blood and venous sodium citrate anticoagulated plasma.

[0112] 3. Sample Collection

[0113] Collect 3 ml of peripheral sodium citrate anticoagulated blood, centrifuge the collected sample at 3000g for 10 minutes, aspirate the plasma and divide it into EP tubes. After the peripheral blood sample collection is completed, use the assessment reagent and reference reagent to detect the content of HBP biomarkers in plasma and peripheral blood respectively and record the test results.

[0114] 4. Statistical Processing

[0115] 4.1 Experimental design

[0116] This experiment was conducted in a randomized, controlled, single-blind manner to ensure that the test results were scientific, rigorous, true and reliable. The selected samples were randomly coded, and the blinded samples were tested. The samples of the same patient were required to be tested on the same day using the assessment system and the reference system. The test data were analyzed using statistical methods to evaluate the results of the heparin-binding protein assay kit (magnetic microparticle chemiluminescence method) prepared according to Example 1 (hereinafter referred to as the assessment system) for detecting different levels of HBP in human peripheral blood and the results of the Zhonghan Shengtai heparin-binding protein assay kit (immunofluorescence dry quantitative method) for detecting different levels of HBP in human plasma (hereinafter referred to as the reference system), and to test whether the results of homologous samples were equivalent.

[0117] 4.2 Analysis method of test data

[0118] (1) Outlier test

[0119] (2) Sample conformity analysis

[0120] The data were organized in a four-cell table, and the sample compliance of the assessment system was evaluated based on the medical decision level of heparin-binding protein (15 ng / mL) of the reference system.

[0121] (3) Consistency analysis

[0122] SPSS 22.0 software was used to analyze the consistency of the experimental data. The method is as follows:

[0123] Overall description: Descriptive statistics are provided for the assessment system and the reference system.

[0124] Absolute bias graph: The difference between the measured values ​​of each sample of the test system and the reference system and the test mean of the two corresponding systems are plotted as a scatter plot. The central horizontal line is 0, and the mean value of the difference ± 1.96 standard deviation is used as the 95% confidence interval line, that is, the consistency limit.

[0125] Relative bias graph: The ratio of the measured values ​​of each sample of the test system and the reference system is plotted as a scatter plot with the test mean of the two corresponding systems. The central horizontal line is 1, and the mean value ± 1.96 standard deviation of the ratio is used as the 95% confidence interval line, i.e., the consistency limit.

[0126] (4) Correlation analysis

[0127] With the test results of the reference system as the horizontal axis and the test results of the assessment system as the vertical axis, a scatter plot was made and the trend line was marked. The uniformity of the data distribution of the scatter plot was visually inspected, and the linear segment was analyzed using SPSS22.0 statistical software to calculate the correlation coefficient r of the test results of the two kits. In principle, r≥0.975 was required.

[0128] (5) Regression analysis

[0129] SPSS22.0 statistical software was used to calculate the regression equation, and variance analysis was performed on the regression equation, and hypothesis testing was performed on the intercept and slope.

[0130] (6) Paired Design Data Test

[0131] SPSS22.0 statistical software was used to perform a normality test on the difference between the measured values ​​of each sample of the assessment system and the reference system. If the test data did not obey the normal distribution, a non-parametric test, the Wilcoxon signed-rank test, was used. If the P value > 0.05, it means that there was no significant difference in the test results of the two kits.

[0132] 4.3 Analysis results of test data

[0133] (1) Outlier test

[0134] Outlier Analysis Methods

[0135] Di=|Yi-Xi|,Di'=|Yi-Xi| / Xi, The absolute detection limit is 4E, and the relative detection limit is 4E'. Compare each Di and Di' with 4E and 4E' respectively, and mark the points that exceed 4E and 4E'. If the above conditions are met at the same time, it is judged as an outlier.

[0136] Table 1: Outlier Observations

[0137] Reference reagents Assessment reagents Absolute Difference Relative Difference Sample No.i Xi Yi Di Di' 1 2 3 4 …… average value / / E E’

[0138] The number of outliers shall not exceed 2.5%, that is, in a clinical trial of 90 samples, no more than 3 outliers shall appear. If it does not exceed the limit, delete the outliers and perform the following data analysis; if it exceeds this limit and no reasonable reason can be found, re-collect samples for data analysis.

[0139] Table 2: Clinical trial results of 90 samples

[0140]

[0141]

[0142]

[0143]

[0144]

[0145] (2) Sample conformity analysis

[0146] A total of 90 homologous peripheral blood and plasma samples were enrolled in this experiment, that is, 90 valid samples were enrolled in the experimental design. The sample compliance of the assessment system was analyzed based on the test results of the reference system. It can be seen that the positive compliance rate is 100.00%, the negative compliance rate is 100%, and the total compliance rate is 100%. The results are shown in Table 3.

[0147] Table 3: Compliance analysis table

[0148]

[0149] Positive coincidence rate = 61 / 61 = 100%

[0150] Negative compliance rate = 29 / 29 = 100%

[0151] Total compliance rate = (61 + 29) / 90 = 100%

[0152] (3) Consistency analysis

[0153] Overall description: Descriptive statistics of the test system (peripheral blood) and reference system (plasma) are shown in Table 4. Statistical software was used to conduct inter-group comparative analysis on the test results of 90 valid samples. Figure 1 It shows that a total of 4 sample results are outside the consistency limit, accounting for 4.44% of the total sample cases; Figure 2 It shows that a total of 4 sample results were outside the consistency limit, accounting for 4.44% of the total sample cases, which means that more than 95% of the samples are within the consistency limit, and that the HBP test results of the assessment system and the reference system are consistent and reach a clinically acceptable level.

[0154] Table 4: Descriptive Statistics

[0155]

[0156] (4) Correlation analysis

[0157] Visual inspection Figure 3 The scatter plots of the two groups of test results show that the data are evenly distributed and change linearly. The linear segment was analyzed using statistical software. The results are shown in Table 5, showing that the assessment system and the reference system are significantly correlated at the 0.01 level, and the correlation coefficient r=1>0.975, which meets the requirements. That is, the correlation between the test results of the assessment system and the reference system is good.

[0158] Table 5: Correlations

[0159]

[0160]

[0161] (5) Regression analysis

[0162] The variance analysis showed that the P value was 0.000<0.05, indicating that the regression analysis was significantly correlated, as shown in Table 6. The linear regression equation of the test results of the assessment system and the reference system was Y=1.002X-0.234, the 95%CI of the intercept -0.234 was (-0.557, -0.089), and the P value was 0.153>0.05, indicating that there was no significant difference between the intercept and the 0 value; the 95%CI of the slope 1.002 was (0.999, 1.005), and the P value was 0.000<0.05, indicating that the linear relationship between the two groups of test results was established. The detailed results are shown in Table 7.

[0163] SPSS22.0 statistical software was used to calculate the regression equation, and variance analysis was performed on the regression equation, and hypothesis testing was performed on the intercept and slope.

[0164] Table 6: ANOVA table

[0165]

[0166] Table 7: Coefficients

[0167]

[0168] (6) Paired Design Data Test

[0169] The difference between the measured values ​​of each sample of the assessment system and the reference system does not conform to the normal distribution, and the results are shown in Table 8. The rank sum test was used to analyze the test results of the two groups, P = 0.056> 0.05, indicating that there is no significant difference between the test results of the assessment system and the reference system, and the results are shown in Table 9. It can be seen that the test results of the assessment system and the reference system are equivalent.

[0170] Table 8: Normality test

[0171]

[0172] The significance level is less than 0.05, and the data do not obey the normal distribution assumption. Because the test data do not obey the normal distribution, the non-parametric test-Wilcoxon signed rank test is used. The P value is greater than 0.05, indicating no significant difference.

[0173] Table 9: Test Statistics

[0174]

[0175]

[0176]

[0177] Although various embodiments of the present invention have been described above, it should be understood that they are provided by way of example only and not by way of limitation. The present invention may be varied and improved without departing from the spirit and scope of the present invention, and these variations and improvements will fall within the scope of the present invention as claimed. The scope of the present invention as claimed is defined by the appended claims and their equivalents.

Claims

1. A heparin binding protein assay kit for peripheral blood, comprising an acridinium ester-labeled heparin binding protein (HBP) antibody, a magnetic particle-labeled HBP antibody, a pre-excitation solution, an excitation solution, an HBP series calibrator, and an HBP series quality control product.

2. The kit according to claim 1, wherein the acridinium ester-labeled HBP antibody is prepared as follows: S1. Take 10 μL of 2.5 mg / ml diluted acridinium ester (NSP-DMAE-NHS) stock solution, add 90 μL of anhydrous DMSO or DMF and dilute 10 times to make 0.25 mg / ml acridinium ester working solution; S2. Dilute 50 μg of HBP antibody to 300 μL with 0.2 M NaHCO3 (pH = 9.0) solution, and add 10 μL of 0.25 mg / ml acridinium ester working solution; S3. Wrap in tin foil and mark at room temperature for 0.5 to 1h; S4. Quench the reaction, add 100 μL of labeling termination buffer (10% lysine 0.2 M NaHCO3 (pH = 9.0) solution), and mix at room temperature for 30 minutes; S5. Purification of the above acridinium ester-conjugated antibody samples 1) Select a 250 ml beaker, inject 150 ml of 0.2 M pH 9.4 carbonate-bicarbonate buffer into it, select a dialysis cassette with a cutoff of 3.5 KDa and take it out of the protective bag, and stand the dialysis cassette at the bottom of the beaker so that the liquid submerges the dialysis cassette membrane; 2) While the dialysis cassette is soaking, the syringe is used to absorb all the acridinium ester-antibody labeling system samples, and the syringe is inverted to expel excess air and pop out bubbles, and then placed aside for use; 3) After the dialysis cassette is soaked, spread absorbent paper on the laboratory table, take out the dialysis cassette, and gently tap the cassette upright and upside down on the absorbent paper to remove excess buffer. The time should be controlled within 1 minute. 4) Slowly insert the needle tip of the syringe into the sampling port on the side of the dialysis box, and the syringe will suck the air in the cavity. Invert the syringe to expel excess air and pop out bubbles; 5) Slowly insert the syringe needle tip into the side sample inlet of the dialysis box, inject 200-500 micrograms of the entire sample, and then suck the air in the cavity with the syringe; 6) Select a 250 ml beaker, add 300-400 times the sample volume of 0.2 M pH 9.4 carbonate-bicarbonate buffer, place the dialysis cassette upright and fully immersed in it, place the small rotor of the magnetic stirrer against the wall of the beaker and put it into the bottom of the beaker, cover the beaker with light-proof paper, turn on the magnetic stirrer, adjust the speed to 150-300 rpm, place the beaker in the center of the magnetic stirrer, and dialyze at room temperature for 0.5 h; 7) Take out the dialysis cassette, discard the buffer in the beaker, add 250-350 times of 0.2M pH 9.4 carbonate-bicarbonate buffer, and repeat step 6) for dialysis at room temperature for 1 hour; 8) Take out the dialysis cassette, discard the buffer in the beaker, add 200-300 times 0.2M pH 9.4 carbonate-bicarbonate buffer, and repeat step 6) for dialysis at room temperature for 2 hours; 9) Take out the dialysis cassette, discard the buffer in the beaker, add 400-600 times 0.2M pH 9.4 carbonate-bicarbonate buffer, and dialyze at 4°C for 12-16 hours; 10) After dialysis is completed, discard the dialysate, take out the dialysis box, insert a new syringe into the sampling port on the other side of the dialysis box, inject appropriate air into it, put the needle in the appropriate position, turn the box to the appropriate position, turn it upside down, and take out the sample with the syringe; 11) First, add 0.5 ml of 0.2 M pH 9.4 carbonate-bicarbonate buffer into the cavity, stir with a magnetic stirrer for 5 min, and then recover; then add 0.3 ml of 0.2 M pH 9.4 carbonate-bicarbonate buffer into the cavity, stir with a magnetic stirrer for 4 min, and then recover; finally, add 0.2 ml of 0.2 M pH 9.4 carbonate-bicarbonate buffer into the cavity, stir with a magnetic stirrer for 3 min, and then recover; 12) Combine all the recovered solutions, mark them as acridinium ester labeled mother solution, and determine the protein concentration.

3. The kit according to claim 1, wherein the magnetic particle-labeled HBP antibody is prepared by an NHS activation step and a protein coupling step, Preferably, NHS activation is performed as follows: 1) After fully mixing the magnetic beads, take 100 μL of the magnetic bead suspension into a 1.5 mL centrifuge tube, place it on a magnetic rack, and remove the supernatant after solid-liquid separation; 2) Take 200 μL of 50 mM, pH 5.0 MES solution, wash the magnetic beads twice, and remove the supernatant; 3) Add 50 μL of freshly prepared EDC solution and 50 μL of NHS solution to the centrifuge tube containing magnetic beads, vortex to mix to fully suspend the magnetic beads, and shake and mix at room temperature for 30 minutes; 4) After the reaction is completed, add 100 μL of the above MES solution and wash twice; Preferably, protein coupling is performed as follows: 1) Place the centrifuge tube in a magnetic separator, remove the supernatant, add 100 μL of protein solution (50 mM MES, pH 5.0) and mix gently; 2) Mix by shaking at room temperature for 2 h, or overnight at 4 °C; 3) After the reaction is completed, the supernatant is removed by magnetic separation, and 100 μL of 50 mM, pH 5.0 MES buffer is added to wash the magnetic beads three times; 4) After the reaction is completed, place the centrifuge tube on a magnetic separation rack, let it stand for 2 minutes, and discard the supernatant; at room temperature, add 100 μL of 50 mM, pH 8.0 ethanolamine solution and rotate and mix for 60 minutes or 50 mM, pH 7.4 Tris buffer and rotate and mix for 15 minutes to block excess active sites; 5) After the reaction, wash 4 times with 100 μL PBS or 50 mM Tris; 6) Add appropriate amount of PBS or Tris preservation solution and store at 4°C; 7) Dilute the antibody-magnetic particle conjugate with a magnetic particle diluent containing 0.05 M Tris, 1% sodium chloride, 2% BSA, 4% trehalose, 0.05% Triton X-100, and 0.5% Proclin 300 to a concentration of 0.5 mg / mL, and store in a 4°C refrigerator.

4. The kit according to any one of claims 1 to 3, wherein the HBP series calibrators and quality control products are prepared as follows: recombinant or natural heparin binding protein is dissolved in a diluent comprising 50 mM pH 7.3 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 0.5% lauryl alcohol polyoxyethylene ether, 50 mM potassium chloride, 2% sucrose, 4% arginine and 0.2% Proclin 300 to prepare calibrators with concentrations of 0, 5, 50, 100, 200, and 300 ng / mL, respectively, and quality control products with concentrations of 8 ng / mL and 40 ng / mL, respectively.

5. The kit according to any one of claims 1 to 3, wherein the pre-stimulation solution comprises 0.1 mol / L HNO3 and 0.1% H2O2, and the stimulation solution comprises 0.25 mol / L NaOH and 2% Triton X-100.

6. The kit according to any one of claims 1 to 3, wherein the kit further comprises a diluent and a buffer.

7. The kit according to claim 6, wherein the diluent for magnetic particle labeling comprises 0.05M Tris, 1% sodium chloride, 2% BSA, 4% trehalose, 0.05% Triton X-100 and 0.5% Proclin300; the diluent for HBP series calibrators and quality control products comprises 50mM pH7.3 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 0.5% polyoxyethylene lauryl alcohol ether, 50mM potassium chloride, 2% sucrose, 4% arginine and 0.2% Proclin300.

8. The kit according to claim 6, wherein the buffer is selected from 0.2 M carbonate-bicarbonate buffer (pH 9.4), MES buffer (50 mM, pH 5.0) and Tris buffer (50 mM, pH 7.4).

9. Use of the kit according to any one of claims 1 to 8 in the preparation of a product for detecting HBP in peripheral blood of a patient.

10. The use according to claim 9, wherein the patient suffers from sepsis or septic shock.

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