Neutrophil gelatinase-associated lipocalin detection kit and preparation method thereof

Abstract

The present invention relates to the technical field of detection kits, and in particular to a detection kit for neutrophil gelatinase-associated lipocalin and a preparation method thereof. The present invention provides a detection kit for neutrophil gelatinase-associated lipocalin, comprising an independently packaged reagent R1 and an independently packaged reagent R2; the reagent R1 comprises an HBR series specific blocker; the reagent R2 comprises carboxyl latex microspheres coupled with NGAL antibodies; the carboxyl content of the carboxyl latex microspheres is greater than 100 μeq / g, and the particle size is 100-400 nm. The present invention uses the HBR series specific blocker as a component of the reagent R1, thereby reducing endogenous interference and avoiding false positive and negative test results of the sample. Furthermore, the present invention selects latex of suitable particle size for coupling with polyantibodies, and optimizes the blocking conditions, which has the advantages of wide linearity, good analytical sensitivity, simple operation, controllable batch difference and controllable cost.

Description

Technical Field

[0001] The invention relates to the technical field of detection kits, in particular to a detection kit for neutrophil gelatinase-associated lipocalin and a preparation method thereof. Background Art

[0002] Neutrophil gelatinase-associated lipocalin (NGAL), also known as human lipocalin 2, has three forms of protein: a monomer with a molecular weight of 25kD, a self-polymerized 46kD homodimer, and a 135kD heterodimer formed with MMP-9 polymers. In recent years, NGAL has been used as a new marker for acute kidney injury (AKI). When diagnosing acute renal injury in the early stage, the NGAL concentration in blood and urine usually increases rapidly, which is most obvious after 2 hours, while traditional indicators such as serum creatinine and urease need to increase significantly after 24 to 72 hours. In addition, NGAL is also a potential new marker for chronic kidney damage. In patients with chronic kidney disease (CKD), NGAL can accurately reflect the degree of kidney damage and is a strong and independent risk indicator for the progression of CKD. In summary, the quantitative detection product of NGAL will have important clinical significance and strong market demand.

[0003] At present, the detection methods for NGAL include immunodiffusion, immunoelectrophoresis, radioimmunoassay, enzyme-linked immunosorbent assay, etc. These methods have the disadvantages of cumbersome operation, special equipment, sample pretreatment, inability to perform batch sample analysis, and inability to be directly tested on a fully automatic biochemical analyzer. Latex immunoturbidimetry is also a commonly used method for determining the concentration of NGAL in human serum and urine. Compared with several other methods, it has the significant characteristics of simple operation, high sensitivity, wide linear range, no pollution, and wide application range. Its basic principle is that the NGAL protein in the clinical sample reacts with the specific anti-human NGAL antibody coupled to the latex microspheres to form an antigen-antibody immune complex, so that the latex microspheres form a network polymer, which increases the turbidity of the detection system. Within a certain range, the NGAL protein concentration is positively correlated with the turbidity. With the increase in clinical testing needs and the popularization of fully automatic biochemical analyzers, the biochemical latex turbidimetry has become the mainstream detection method in clinical practice due to its advantages of short reaction time, good precision, and easy automation.

[0004] During the use of in vitro diagnostic reagents, any immunoassay method may be interfered with, resulting in false positive or false negative results. Endogenous interference is often caused by cross-reaction between the antibodies used in the reagents and endogenous substances in the patient's samples. This type of interference is collectively referred to as endogenous interference. Endogenous interference is a phenomenon that cannot be avoided in immunoassays. Eliminating interference is an important task in the development of immunoassay kits. Various commercially available NGAL latex immunoturbidimetric test kits also have this problem. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a detection kit for neutrophil gelatinase-associated lipocalin and a preparation method thereof. The reagent R1 in the detection kit provided by the present invention contains a suitable active blocker, thereby reducing endogenous interference and avoiding false positive or negative test results of the sample.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a neutrophil gelatinase-associated lipocalin detection kit, comprising an independently packaged reagent R1 and an independently packaged reagent R2; the reagent R1 comprises a buffer, a surfactant, a coagulant, a blocker, a preservative, a stabilizer and an electrolyte; the blocker is one or more of the HBR series specific blockers, and the concentration of the blocker in the reagent R1 is 5-20 g / L;

[0008] The reagent R2 comprises carboxyl latex microspheres coupled with NGAL antibodies, a buffer, an electrolyte, a surfactant, a stabilizer and a preservative; the carboxyl content of the carboxyl latex microspheres is greater than 100 μeq / g, and the particle size is 100-400 nm; when preparing the carboxyl latex microspheres coupled with NGAL antibodies, the blocking agent is a glycine blocking solution and a Blockmaster CE series blocking agent.

[0009] Preferably, the concentration of the glycine blocking solution is 0.5-2 mol / L.

[0010] Preferably, in the reagent R1, the buffer comprises one or more of phosphate buffer, HEPES buffer, Tris buffer and MOPSO buffer, and the pH value is 6.0-9.0;

[0011] The coagulant is one or more of polyethylene glycol 6000, polyethylene glycol 8000 or polyethylene glycol 35000, and the concentration of the coagulant in the reagent R1 is 10-50 g / L;

[0012] In the reagent R1, the electrolyte includes one or more of sodium chloride, potassium chloride and calcium chloride, and the concentration of the electrolyte in the reagent R1 is 5-50 g / L;

[0013] In the reagent R1, the stabilizer includes one or more of mannitol, bovine serum albumin and trehalose, and the concentration of the stabilizer in the reagent R1 is 5-50 g / L;

[0014] In the reagent R1, the surfactant includes one or more of Tween 20, Tween 40 and TX-100, with a concentration of 5 to 50 g / L;

[0015] In the reagent R1, the preservative is sodium azide and / or Proclin 300, and the concentration of the preservative in the reagent R1 is 0.5-20 g / L.

[0016] Preferably, in the reagent R2, the buffer comprises one or more of phosphate buffer, glycine buffer, MES buffer, HEPES buffer, Tris buffer and MOPSO buffer, and the pH value is 6.0-9.0;

[0017] In the reagent R2, the electrolyte includes one or more of sodium chloride, potassium chloride and calcium chloride, and the concentration of the electrolyte in the reagent R2 is 5-50 g / L;

[0018] In the reagent R2, the stabilizer includes one or more of mannitol, bovine serum albumin and trehalose, and the concentration of the stabilizer in the reagent R2 is 5-50 g / L;

[0019] In the reagent R2, the surfactant includes Tween 20 at a concentration of 5 to 50 g / L;

[0020] In the reagent R2, the preservative is sodium azide and / or Proclin 300, and the concentration of the preservative in the reagent R1 is 0.5-20 g / L.

[0021] Preferably, the volume ratio of the reagent R1 to the reagent R2 is 3:1.

[0022] Preferably, the detection kit further comprises a calibrator and a quality control product; the calibrator comprises a recombinant or natural NGAL protein; the quality control product comprises a recombinant or natural NGAL protein.

[0023] The present invention provides a method for preparing the detection kit described in the above technical solution, comprising the following steps:

[0024] Mixing the components in reagent R1 according to the proportion to obtain the reagent R1;

[0025] The buffer solution, electrolyte, surfactant, stabilizer and preservative in reagent R2 are mixed in proportion to obtain R2 preservation solution;

[0026] Carboxyl latex microspheres and 1-ethyl-(3-dimethylaminopropyl)carbodiimide are activated to obtain a first reaction mixture;

[0027] Cross-linking the first reaction mixture and the NGAL antibody to obtain a second reaction mixture;

[0028] Performing a first blocking operation on the second reaction mixture and the glycine blocking solution to obtain a third reaction mixture;

[0029] Performing a second blocking on the third reaction mixture and Blockmaster CE series blocking agent to obtain a fourth reaction mixture;

[0030] Centrifuging the fourth reaction mixture, removing the supernatant, and obtaining the precipitate as the carboxyl latex microspheres coupled with the NGAL antibody;

[0031] The carboxyl latex microspheres coupled with the NGAL antibody are mixed with the R2 preservation solution in proportion to obtain the reagent R2.

[0032] Preferably, the temperature of the first reaction is 25~30°C, and the time is 15~20 min; the temperature of the second reaction is 25~30°C, and the time is 60~150 min; the temperature of the third reaction is 25~30°C, and the time is 30~90 min; the temperature of the fourth reaction is 25~30°C, and the time is 30~90 min.

[0033] Preferably, the mass ratio of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide to the carboxyl latex microspheres is 1:40-60; the mass ratio of the NGAL antibody to the carboxyl latex microspheres is 1:40-60; the concentration of the glycine blocking solution in the first closed system is 1-10wt%; and the concentration of the Blockmaster CE series blocking agent in the second closed system is 0.5-5wt%.

[0034] Preferably, the fourth reaction mixture is centrifuged at a speed of 15000-25000 rpm; and the method of mixing the carboxyl latex microspheres coupled with the NGAL antibody with the R2 preservation solution comprises ultrasonic resuspension.

[0035] Beneficial effects:

[0036] The present invention provides a detection kit for neutrophil gelatinase-associated lipocalin, comprising an independently packaged reagent R1 and an independently packaged reagent R2; the reagent R1 comprises a buffer, a surfactant, a coagulant, a blocker, a preservative, a stabilizer and an electrolyte; the blocker is one or more of the HBR series specific blockers, and the concentration of the blocker in the reagent R1 is 5-20 g / L; the reagent R2 comprises carboxyl latex microspheres coupled with NGAL antibodies, a buffer, an electrolyte, a surfactant, a stabilizer and a preservative; the carboxyl content of the carboxyl latex microspheres is greater than 100 μeq / g, and the particle size is 100-400 nm; when preparing the carboxyl latex microspheres coupled with NGAL antibodies, the blocking agent is a glycine blocking solution and a Blockmaster CE series blocking agent. The detection kit provided by the present invention is a latex immunoturbidimetric detection kit with good anti-interference performance, high sensitivity and capable of simultaneously detecting neutrophil gelatinase-associated lipocalin in serum and urine. The present invention uses HBR series specific blocking agents as components of reagent R1, thereby reducing endogenous interference, improving the stability of the performance of the antibody after binding to latex particles, avoiding false positive and negative test results of the sample, and has the advantages of strong specificity, high sensitivity, good stability of the kit, and can efficiently detect the NGAL content in urine and serum and has good sample dilution test results.

[0037] Furthermore, the present invention selects latex with a suitable particle size for coupling with polyantibodies and optimizes the blocking conditions, which has the advantages of wide linearity, good analytical sensitivity, simple operation, controllable batch difference and controllable cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0039] Figure 1 This is the comparison result of test case 1;

[0040] Figure 2 This is the comparison result of test example 2;

[0041] Figure 3 This is the comparison result of test case 3;

[0042] Figure 4 The linearity test result of the test kit described in Example 1;

[0043] Figure 5 This is the linearity test result of the test kit described in Comparative Example 1;

[0044] Figure 6 This is the linearity test result of the test kit described in Comparative Example 3;

[0045] Figure 7 This is the linearity test result of the test kit described in Comparative Example 4;

[0046] Figure 8 The results of serum sample correlation test of the test kit described in Example 1;

[0047] Fig. 9 This is the urine sample correlation test result of the test kit described in Example 1. DETAILED DESCRIPTION

[0048] The present invention provides a neutrophil gelatinase-associated lipocalin detection kit, comprising an independently packaged reagent R1 and an independently packaged reagent R2; the reagent R1 comprises a buffer, a surfactant, a coagulant, a blocker, a preservative, a stabilizer and an electrolyte; the blocker is one or more of the HBR series specific blockers, and the concentration of the blocker in the reagent R1 is 5-20 g / L;

[0049] The reagent R2 comprises carboxyl latex microspheres coupled with NGAL antibodies, a buffer, an electrolyte, a surfactant, a stabilizer and a preservative; the carboxyl content of the carboxyl latex microspheres is greater than 100 μeq / g, and the particle size is 100-400 nm; when preparing the carboxyl latex microspheres coupled with NGAL antibodies, the blocking agent is a glycine blocking solution and a Blockmaster CE series blocking agent.

[0050] The reagent R1 of the present invention includes a buffer, a surfactant, a coagulant, a blocker, a preservative, a stabilizer and an electrolyte; the blocker is one or more of the HBR series specific blockers, and the concentration of the blocker in the reagent R1 is 5-20 g / L. As an embodiment, the blocker is an HBR active blocker, and the concentration of the blocker in the reagent R1 can be 8-15 g / L; as another embodiment, the concentration of the blocker in the reagent R1 can be 10 g / L. The present invention uses the HBR series specific blocker as a component of the reagent R1, thereby reducing endogenous interference, improving the stability of the performance of the antibody after binding to latex particles, avoiding false positive and negative test results of the sample, and has the advantages of strong specificity, high sensitivity, good stability of the kit, and can efficiently detect the NGAL content in urine and serum and good sample dilution test results.

[0051] As an embodiment, in the reagent R1, the buffer includes one or more of phosphate buffer, HEPES buffer, Tris buffer and MOPSO buffer, and the pH value is 6.0~9.0; as an embodiment, the buffer can be MOPSO buffer, and the pH value can be any value such as 6.0, 7.0, 7.4, 8.0 or 9.0.

[0052] As an embodiment, the coagulant is one or more of polyethylene glycol 6000, polyethylene glycol 8000 or polyethylene glycol 35000, and the concentration of the coagulant in reagent R1 is 10~50g / L; as an embodiment, the coagulant can be polyethylene glycol 6000, and the concentration of polyethylene glycol 6000 in the reagent R1 can be 20g / L.

[0053] As an embodiment, in the reagent R1, the electrolyte includes one or more of sodium chloride, potassium chloride and calcium chloride, and the concentration of the electrolyte in the reagent R1 is 5~50g / L; as an embodiment, in the reagent R1, the electrolyte can be sodium chloride, and the concentration of sodium chloride in the reagent R1 is 10g / L.

[0054] As an embodiment, in the reagent R1, the stabilizer includes one or more of mannitol, bovine serum albumin and trehalose, and the concentration of the stabilizer in the reagent R1 is 5~50g / L; as an embodiment, in the reagent R1, the stabilizer can be bovine serum albumin and trehalose; as an embodiment, in the reagent R1, the concentration of the stabilizer in the reagent R1 is 20g / L.

[0055] As an embodiment, in the reagent R1, the surfactant may be one or more of Tween 20, Tween 40 and TX-100, with a concentration of 5 to 50 g / L; as an embodiment, in the reagent R1, the concentration of the surfactant may be 10 g / L.

[0056] As an embodiment, in the reagent R1, the preservative is sodium azide and / or Proclin300, and the concentration of the preservative in the reagent R1 is 0.5~20g / L; as an embodiment, in the reagent R1, the preservative may be sodium azide, and the concentration of sodium azide in the reagent R1 is 1g / L.

[0057] The reagent R2 of the present invention comprises carboxyl latex microspheres coupled with NGAL antibodies, a buffer, an electrolyte, a surfactant, a stabilizer and a preservative; the carboxyl content of the carboxyl latex microspheres is greater than 100 μeq / g, and the particle size is 100-400 nm; when preparing the carboxyl latex microspheres coupled with NGAL antibodies, the blocking agent is a glycine blocking solution and a Blockmaster CE series blocking agent. As an embodiment, the NGAL antibody can be a specific anti-human NGAL antibody; as another embodiment, the NGAL antibody can be a rabbit anti-human polyclonal antibody and / or a sheep anti-human polyclonal antibody. As an embodiment, the Blockmaster CE series blocking agent can be a CE510 blocking agent; as an embodiment, the concentration of the glycine blocking solution is 0.5-2 mol / L.

[0058] As an embodiment, the particle size of the carboxyl latex microspheres is 100-250 nm, and the carboxyl content is greater than 100 μeq / g and less than or equal to 200 μeq / g; as another embodiment, the particle size of the carboxyl latex microspheres can be 120 nm, and the carboxyl content can be 150 μeq / g. In order to balance analytical sensitivity and linearity, various commercially available NGAL reagents adopt means such as mixing large and small balls, modifying antibodies and latex, introducing more uncertain factors and increasing costs. The present invention selects latex of suitable particle size for coupling with multiple antibodies, and optimizes the blocking conditions, which has the advantages of wide linearity, good analytical sensitivity, simple operation, controllable batch difference, and controllable cost.

[0059] As an embodiment, in the reagent R2, the buffer includes one or more of phosphate buffer, glycine buffer, MES buffer, HEPES buffer, Tris buffer and MOPSO buffer, and the pH value is 6.0~9.0; as an embodiment, the buffer can be Tris buffer, and the pH value can be any value such as 6.0, 7.0, 7.4, 8.0 or 9.0.

[0060] As an embodiment, in the reagent R2, the electrolyte includes one or more of sodium chloride, potassium chloride and calcium chloride, and the concentration of the electrolyte in the reagent R2 is 5~50g / L; as an embodiment, in the reagent R2, the electrolyte can be sodium chloride, and the concentration of sodium chloride in the reagent R2 is 9.5g / L.

[0061] As an embodiment, in the reagent R2, the stabilizer includes one or more of mannitol, bovine serum albumin and trehalose, and the concentration of the stabilizer in the reagent R2 is 5~50g / L; as an embodiment, in the reagent R2, the stabilizer may be bovine serum albumin and trehalose; as an embodiment, in the reagent R2, the concentration of the stabilizer in the reagent R2 is 21.5g / L.

[0062] As an embodiment, in the reagent R2, the surfactant may be one or more of Tween 20, Tween 40 and TX-100, with a concentration of 5 to 50 g / L; as an embodiment, in the reagent R2, the concentration of the surfactant may be 2 g / L.

[0063] As an embodiment, in the reagent R2, the preservative is sodium azide and / or Proclin300, and the concentration of the preservative in the reagent R1 is 0.5~20g / L; as an embodiment, in the reagent R2, the preservative may be sodium azide, and the concentration of sodium azide in the reagent R2 is 1g / L.

[0064] As an embodiment, the volume ratio of the reagent R1 to the reagent R2 is 3:1.

[0065] As an embodiment, the detection kit further comprises a calibrator and a quality control product; the calibrator comprises a recombinant or natural NGAL protein; the quality control product comprises a recombinant or natural NGAL protein.

[0066] The present invention provides a method for preparing the detection kit described in the above technical solution, comprising the following steps:

[0067] Mixing the components in reagent R1 according to the proportion to obtain the reagent R1;

[0068] The buffer solution, electrolyte, surfactant, stabilizer and preservative in reagent R2 are mixed in proportion to obtain R2 preservation solution;

[0069] Carboxyl latex microspheres and 1-ethyl-(3-dimethylaminopropyl)carbodiimide are activated to obtain a first reaction mixture;

[0070] Cross-linking the first reaction mixture and the NGAL antibody to obtain a second reaction mixture;

[0071] Performing a first blocking operation on the second reaction mixture and the glycine blocking solution to obtain a third reaction mixture;

[0072] Performing a second blocking on the third reaction mixture and Blockmaster CE series blocking agent to obtain a fourth reaction mixture;

[0073] Centrifuging the fourth reaction mixture, removing the supernatant, and obtaining the precipitate as the carboxyl latex microspheres coupled with the NGAL antibody;

[0074] The carboxyl latex microspheres coupled with the NGAL antibody are mixed with the R2 preservation solution in proportion to obtain the reagent R2.

[0075] As an embodiment, the temperature of the first reaction is 25~30°C, and the time is 15~20min; the temperature of the second reaction is 25~30°C, and the time is 60~150min; the temperature of the third reaction is 25~30°C, and the time is 30~90min; the temperature of the fourth reaction is 25~30°C, and the time is 30~90min.

[0076] As an embodiment, the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to carboxyl latex microspheres is 1:40~60; the mass ratio of NGAL antibody to carboxyl latex microspheres is 1:40~60; the concentration of the glycine blocking solution in the first closed system is 1~10wt%; the concentration of the Blockmaster CE series blocking agent in the second closed system is 0.5~5wt%.

[0077] As an embodiment, the fourth reaction mixture is centrifuged at a speed of 15000-25000 rpm; and the method of mixing the carboxyl latex microspheres coupled with the NGAL antibody with the R2 preservation solution includes ultrasonic resuspension.

[0078] To further illustrate the present invention, the detection kit for neutrophil gelatinase-associated lipocalin and the preparation method thereof provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0079] The HBR blocker, carboxyl latex microspheres, NGAL rabbit anti-human polyclonal antibody, and NGAL recombinant protein used in the present invention are all purchased externally, and the sources are:

[0080] Blocker HBR-6: Hangzhou Boyue Biotechnology Co., Ltd.

[0081] Carboxyl latex microspheres: Suzhou Weidu Biotechnology Co., Ltd., product number PS0120CHA (carboxyl content > 100 μeq / g) and PS0120CLA (carboxyl content < 100 μeq / g).

[0082] NGAL rabbit anti-human polyclonal antibody: Guilin Yingmite Biotechnology Co., Ltd., catalog number KAT0028.R

[0083] NGAL recombinant protein: Guilin Yingmeite Biotechnology Co., Ltd., catalog number KAY0028.C

[0084] Example 1

[0085] A neutrophil gelatinase-associated lipocalin (NGAL) detection kit, comprising independently packaged reagent R1, reagent R2, calibrator and quality control product; the volume ratio of the reagent R1 to the reagent R2 is 3:1;

[0086] The reagent R1 is composed of the following components in the following concentrations: 3-(N-morpholinyl)-2-hydroxypropanesulfonic acid (MOPSO) 11.3 g / L, sodium chloride 10.0 g / L, trehalose 10.0 g / L, polyethylene glycol 6000 (PEG-6000) 20.0 g / L, Tween 20 10.0 g / L, bovine serum albumin (BSA) 10.0 g / L, blocking agent HBR-6 10.0 g / L and NaN3 1.0 g / L; the solvent of the reagent R1 is water, and the pH value of the reagent R1 is 7.4; the reagent R1 is prepared according to the above-mentioned component concentrations and then filtered through a 0.24 μm filter membrane;

[0087] The preparation method of the reagent R2 is as follows:

[0088] The latex particle precipitate coupled with NGAL rabbit anti-human polyclonal antibody is ultrasonically resuspended to 1wt% by using R2 preservation solution to obtain the reagent R2; the R2 preservation solution is composed of the following components in concentration: Tris (Tris) 12.1g / L, sodium chloride 9.5g / L, trehalose 20.0g / L, Tween 20 2.0g / L, BSA 1.5g / L and NaN31.0g / L; the solvent of the reagent R2 is water, and the pH value of the R2 preservation solution is 7.4; the R2 preservation solution is prepared according to the concentrations of the above components and then filtered through a 0.24μm filter membrane;

[0089] The preparation method of the latex particle precipitation coupled with NGAL rabbit anti-human polyclonal antibody is as follows:

[0090] 1) diluting carboxyl latex microspheres to 1 wt% with 50 mM Tris and mixing with 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), reacting at 25° C. for 20 min to obtain a first reaction mixture; the particle size of the carboxyl latex microspheres is 120 nm, and the carboxyl content is greater than 100 μeq / g; the mass ratio of the EDC to the carboxyl latex microspheres is 1:50;

[0091] 2) mixing the first reaction mixture and NGAL rabbit anti-human polyclonal antibody, reacting at 25° C. for 60 min to obtain a second reaction mixture; the mass ratio of the NGAL rabbit anti-human polyclonal antibody to the carboxyl latex microspheres in the first reaction mixture is 1:60;

[0092] 3) adding 2 M glycine blocking solution to the second reaction mixture to a final concentration of 5 wt % in the system, reacting at 25° C. for 60 min to obtain a third reaction mixture;

[0093] 4) adding CE510 blocking agent to the third reaction mixture to a final concentration of 1 wt % in the system, reacting at 25° C. for 60 min to obtain a fourth reaction mixture;

[0094] 5) centrifuging the fourth reaction mixture at 4° C. and 20,000 rpm, removing the supernatant, and obtaining a precipitate of latex particles coupled with NGAL rabbit anti-human polyclonal antibodies;

[0095] The preparation method of the calibrator is as follows:

[0096] 10mM TRIS buffer (pH7.40), 0.5wt% bovine serum albumin (BSA), 0.5wt% Tween-20, 0.1wt% sodium azide and NGAL recombinant protein, wherein the concentrations of NGAL recombinant protein are 150ng / mL, 600ng / mL, 1250ng / mL, 2500ng / mL and 5000ng / mL respectively.

[0097] The preparation method of the quality control product is as follows:

[0098] 10 mM TRIS buffer (pH 7.40), 0.5 wt% bovine serum albumin (BSA), 0.5 wt% Tween-20, 0.1 wt% sodium azide and NGAL recombinant protein, wherein the concentrations of NGAL recombinant protein are 200 ng / mL and 800 ng / mL, respectively.

[0099] Comparative Example 1

[0100] A detection kit similar to Example 1, except that the reagent R1 does not contain the blocking agent HBR-6.

[0101] Comparative Example 2

[0102] A detection kit similar to Example 1, except that the carboxyl content of the carboxyl latex microspheres is ≤100 μeq / g.

[0103] Comparative Example 3

[0104] A detection kit similar to Example 1, the only difference being that the reaction in step 3) is not performed, and the third reaction mixture in step 4) is replaced by the second reaction mixture.

[0105] Comparative Example 4

[0106] A detection kit similar to Example 1, except that the reaction in step 3) is not performed, the third reaction mixture in step 4) is replaced by the second reaction mixture, and the CE510 blocking agent is replaced by BSA with a volume concentration of 10%.

[0107] Example 2

[0108] A detection kit similar to Example 1, except that the concentration of the blocking agent HBR-6 in the reagent R1 is 5 g / L.

[0109] Example 3

[0110] A detection kit similar to Example 1, except that the concentration of the blocking agent HBR-6 in the reagent R1 is 20 g / L.

[0111] Example 4 Reagent Detection Process

[0112] The measurement parameters are shown in Table 1.

[0113] Table 1 Measurement parameters

[0114]

[0115] The determination method is: mix 150μL reagent R1 and 3μL sample, incubate at 37℃ for 5 minutes, add 50μL reagent R2, mix well, incubate for 20 seconds and read the absorbance A1, incubate at 37℃ for 5 minutes and read the absorbance A2, and calculate ∆A=A2-A1.

[0116] Test Example 1

[0117] According to the detection method of Example 4, calibration was performed on the fully automatic biochemical analyzer AU680 using NGAL calibrators of different concentrations (150 ng / mL, 600 ng / mL, 1250 ng / mL, 2500 ng / mL, 5000 ng / mL) in the detection kit to determine the linear relationship of the detection kits of Example 1 (R1 with blocker) and Comparative Example 1 (R1 without blocker). The results are as follows: Figure 1 As shown in the figure, it can be seen that the linear relationship between R1 and △A value is better when there is a blocker, the slope of the fitted standard curve will be larger, and the accuracy and sensitivity will be greatly improved.

[0118] Test Example 2

[0119] The detection method of Example 4 was used to calibrate the NGAL calibrators of different concentrations (150 ng / mL, 600 ng / mL, 1250 ng / mL, 2500 ng / mL, 5000 ng / mL) in the detection kit on the fully automatic biochemical analyzer AU680, and the linear relationship of the detection kits of Example 1 (high carboxyl) and Comparative Example 2 (low carboxyl) was determined. The results are as follows: Figure 2 As shown in the figure, it can be seen that the linear relationship between the high carboxyl group reagent and the △A value is better, and the △A value is generally higher than that of the low carboxyl group reagent. The slope of the fitted standard curve will be larger, and the sensitivity and linear high-end measurement performance will be greatly improved.

[0120] Test Example 3

[0121] According to the detection method of Example 4, calibration was performed on the fully automatic biochemical analyzer AU680 using NGAL calibrators of different concentrations (150 ng / mL, 600 ng / mL, 1250 ng / mL, 2500 ng / mL, 5000 ng / mL) in the detection kit to determine the linear relationship of the detection kits of Example 1 (two blocking agents), Comparative Example 3 (single CE510 blocking) and Comparative Example 4 (single BSA blocking). The results are as follows: Figure 3 As shown in the figure, it can be seen that the linear relationship between the double blocking reagent group and the △A value is the best, and the slope of the fitted standard curve is appropriate; the △A value of the single CE510 blocking reagent group is overall skew, and the slope of the fitted standard curve is the smallest; the △A value of the single 10% BSA blocking reagent group is low at the low end and high at the high end, which will lead to high values ​​of low-end samples and may cause false positives.

[0122] Test Example 4 Blank Limit Detection

[0123] The detection kits described in Example 1, Comparative Example 1, Comparative Example 3 and Comparative Example 4 were used to detect blank samples (purified water) according to the detection method of Example 4, and the test was repeated 20 times. The average value M and standard deviation SD of the 20 test results were calculated, and the M+2SD value was calculated as the blank limit result. The results are shown in Tables 2 and 3.

[0124] Table 2 Results of the test kits of Example 1 and Comparative Example 1 (unit of test value: ng / mL)

[0125]

[0126] Table 3 Results of the detection kits of Comparative Example 3 and Comparative Example 4 (detection value unit: ng / mL)

[0127]

[0128] The blank limit reflects the background noise level of the detection system in the absence of a sample. The blank limit can be used to evaluate the sensitivity and accuracy of the detection method. As shown in Tables 2 and 3, the blank limit is significantly reduced when a blocking agent is added to reagent R1, thereby improving the reliability of the detection results.

[0129] Test Example 5 Linear Detection

[0130] Use 0.9% saline to dilute the high-concentration sample close to the upper limit of the linear interval (i.e., the 5000 ng / mL NGAL calibrator in the kit of Example 1), and dilute a total of 9 gradient samples: 19.5 ng / mL, 39.1 ng / mL, 78.1 ng / mL, 156.3 ng / mL, 312.5 ng / mL, 625 ng / mL, 1250 ng / mL, 2500 ng / mL, 5000 ng / mL. Use the detection kits described in Example 1, Comparative Example 1, Comparative Example 3 and Comparative Example 4 to detect the above samples according to the detection method of Example 4, and test each dilution 3 times to calculate the mean (Yi) of the detection results for each dilution. Take the dilution (Xi) as the independent variable and Yi as the dependent variable to obtain the linear regression equation. Calculate the correlation coefficient (r) of the linear regression to determine whether the result meets the requirements. Substitute Xi into the linear regression equation, calculate the estimated value of Yi and the relative deviation or absolute deviation of Yi from its estimated value, and the linear correlation results are shown in Table 1. Figure 4~Figure 7 .

[0131] The results show that the best result is obtained with R1 containing a blocking agent and R2 containing a double blocking agent (i.e., the kit of Example 1). 2 =1, the maximum absolute deviation in the range of ≤200 ng / mL is 2.44 ng / mL, and the maximum relative deviation in the range of (200-5000] ng / mL is 3.1%.

[0132] Results of R1 without blocking agent and R2 with double blocking agent (i.e., the kit of Comparative Example 1): R 2 =0.9936, the maximum absolute deviation in the range of ≤200 ng / mL was 19.92 ng / mL, and the maximum relative deviation in the range of (200-5000] ng / mL was 13.4%.

[0133] Results of R1 with blocker plus R2 blocked with CE510 (i.e., the kit of Comparative Example 3): R 2 =0.9994, the maximum absolute deviation in the range of ≤200 ng / mL is 5.83 ng / mL, and the maximum relative deviation in the range of (200-5000] ng / mL is -4.0%.

[0134] Results of R1 with blocking agent plus R2 blocked with 10% BSA (i.e., the kit of Comparative Example 4): R2 =0.9996, the maximum absolute deviation in the range of ≤200 ng / mL is 13.47 ng / mL, and the maximum relative deviation in the range of (200-5000] ng / mL is 13.4%.

[0135] Test Example 6: Anti-endogenous interference detection

[0136] Various immunoassay methods may be subject to exogenous or endogenous interference and produce false positive or false negative results. Endogenous interference is often caused by the reaction between the antibodies used in the reagents and the endogenous substances in the sample. The present invention determines the relative deviation between the measured value result after the sample is diluted 2 times and the measured value result of the original sample. The details are as follows:

[0137] The detection kits described in Example 1, Comparative Example 1, Example 2 and Example 3 were used to detect serum samples collected from 20 hospitals and their double dilutions according to the detection method of Example 4. The results are shown in Table 4.

[0138] Table 4 Test results of different test reagents and different samples (unit: ng / mL)

[0139]

[0140] From the results, it can be seen that the relative deviation of the measured results of the samples after double dilution of the non-blocker reagent group and the measured results of the original samples (average relative deviation 6.5%) is significantly greater than that of the blocker reagent group: the average relative deviation of Example 2 with 5g / L blocker is 5.0%, the average relative deviation of Example 1 with 10g / L blocker is 2.9%, and the average relative deviation of Example 2 with 20g / L blocker is 4.3%. The addition of blockers effectively eliminates endogenous interference, improves the reliability of the test results, and avoids false positive or false negative results.

[0141] Test Example 7: Sample Correlation Detection

[0142] The detection kit prepared in Example 1 of the present invention and the control reagent (Beijing Leadman) were used to measure 50 serum samples and 50 urine samples collected from the hospital, and the measured values ​​were subjected to correlation analysis. The measurement results are shown in Tables 5 to 6 and Figure 8~Figure 9 The results showed that the correlation between the present invention and the control reagent was very high. 2 ≥0.997,.

[0143] Table 5 Detection results of different serum samples by different reagents and correlation analysis results

[0144]

[0145] Table 6 Detection results of different urine samples by different reagents and correlation analysis results

[0146]

[0147] In summary, the detection kit provided by the present invention has the advantages of strong specificity, high sensitivity and good stability when detecting neutrophil gelatinase-associated lipocalin, and can efficiently detect the NGAL content in urine and serum.

[0148] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A detection kit for neutrophil gelatinase-associated lipocalin, characterized in that: It is composed of an independently packaged reagent R1 and an independently packaged reagent R2; the reagent R1 is a buffer, a surfactant, a coagulant, a blocker, a preservative, a stabilizer and an electrolyte; the blocker is one or more of the HBR series of specific blockers, and the concentration of the blocker in the reagent R1 is 5-20 g / L; The reagent R2 is carboxyl latex microspheres coupled with NGAL antibody, a buffer, an electrolyte, a surfactant, a stabilizer and a preservative; the carboxyl content of the carboxyl latex microspheres is greater than 100 μeq / g, and the particle size is 100-400 nm; The method for preparing the carboxyl latex microspheres coupled with the NGAL antibody is as follows: subjecting the carboxyl latex microspheres to an activation reaction with 1-ethyl-(3-dimethylaminopropyl)carbodiimide to obtain a first reaction mixture; subjecting the first reaction mixture to a cross-linking reaction with the NGAL antibody to obtain a second reaction mixture; subjecting the second reaction mixture to a first blocking reaction with a glycine blocking solution to obtain a third reaction mixture; subjecting the third reaction mixture to a BlockmasterCE series blocking agent to a second blocking reaction to obtain a fourth reaction mixture; and centrifuging the fourth reaction mixture to remove the supernatant to obtain a precipitate of the carboxyl latex microspheres coupled with the NGAL antibody.

2. The detection kit according to claim 1, characterized in that The concentration of the glycine blocking solution is 0.5-2 mol / L.

3. The detection kit according to claim 1, characterized in that In the reagent R1, the buffer includes one or more of phosphate buffer, HEPES buffer, Tris buffer and MOPSO buffer, and the pH value is 6.0-9.0; The coagulant is one or more of polyethylene glycol 6000, polyethylene glycol 8000 or polyethylene glycol 35000, and the concentration of the coagulant in the reagent R1 is 10-50 g / L; In the reagent R1, the electrolyte includes one or more of sodium chloride, potassium chloride and calcium chloride, and the concentration of the electrolyte in the reagent R1 is 5-50 g / L; In the reagent R1, the stabilizer includes one or more of mannitol, bovine serum albumin and trehalose, and the concentration of the stabilizer in the reagent R1 is 5-50 g / L; In the reagent R1, the surfactant includes one or more of Tween 20, Tween 40 and TX-100, with a concentration of 5 to 50 g / L; In the reagent R1, the preservative is sodium azide and / or Proclin 300, and the concentration of the preservative in the reagent R1 is 0.5-20 g / L.

4. The detection kit according to claim 1, characterized in that In the reagent R2, the buffer includes one or more of phosphate buffer, glycine buffer, MES buffer, HEPES buffer, Tris buffer and MOPSO buffer, and the pH value is 6.0-9.0; In the reagent R2, the electrolyte includes one or more of sodium chloride, potassium chloride and calcium chloride, and the concentration of the electrolyte in the reagent R2 is 5-50 g / L; In the reagent R2, the stabilizer includes one or more of mannitol, bovine serum albumin and trehalose, and the concentration of the stabilizer in the reagent R2 is 5-50 g / L; In the reagent R2, the surfactant includes Tween 20 at a concentration of 5 to 50 g / L; In the reagent R2, the preservative is sodium azide and / or Proclin 300, and the concentration of the preservative in the reagent R1 is 0.5-20 g / L.

5. The detection kit according to claim 1, characterized in that The volume ratio of the reagent R1 to the reagent R2 is 3:

1.

6. The detection kit according to claim 1, characterized in that The detection kit also includes a calibrator and a quality control product; the calibrator includes a recombinant or natural NGAL protein; the quality control product includes a recombinant or natural NGAL protein.

7. The method for preparing the detection kit according to any one of claims 1 to 6, characterized in that: The following steps are involved: Mixing the components in reagent R1 according to the proportion to obtain the reagent R1; The buffer solution, electrolyte, surfactant, stabilizer and preservative in reagent R2 are mixed in proportion to obtain R2 preservation solution; Carboxyl latex microspheres and 1-ethyl-(3-dimethylaminopropyl)carbodiimide are activated to obtain a first reaction mixture; Cross-linking the first reaction mixture and the NGAL antibody to obtain a second reaction mixture; Performing a first blocking operation on the second reaction mixture and the glycine blocking solution to obtain a third reaction mixture; Performing a second blocking on the third reaction mixture and the BlockmasterCE series blocking agent to obtain a fourth reaction mixture; Centrifuging the fourth reaction mixture, removing the supernatant, and obtaining the precipitate as the carboxyl latex microspheres coupled with the NGAL antibody; The carboxyl latex microspheres coupled with the NGAL antibody are mixed with the R2 preservation solution in proportion to obtain the reagent R2.

8. The preparation method according to claim 7, characterized in that: The temperature of the first reaction is 25~30℃, and the time is 15~20min; the temperature of the second reaction is 25~30℃, and the time is 60~150min; the temperature of the third reaction is 25~30℃, and the time is 30~90min; the temperature of the fourth reaction is 25~30℃, and the time is 30~90min.

9. The preparation method according to claim 7, characterized in that: The mass ratio of the 1-ethyl-(3-dimethylaminopropyl) carbodiimide to the carboxyl latex microspheres is 1:40-60; the mass ratio of the NGAL antibody to the carboxyl latex microspheres is 1:40-60; the concentration of the glycine blocking solution in the first closed system is 1-10wt%; the concentration of the BlockmasterCE series blocking agent in the second closed system is 0.5-5wt%.

10. The preparation method according to claim 7, characterized in that: The rotation speed of the fourth reaction mixture centrifugation is 15000-25000 rpm; the method of mixing the carboxyl latex microspheres coupled with NGAL antibody and the R2 preservation solution includes ultrasonic resuspension.

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