An anti-ngal monoclonal antibody complex fluorescent microsphere complex, a preparation method thereof, and an ngal detection kit

Abstract

The application relates to the technical field of in-vitro reagent detection, in particular to an anti-NGAL monoclonal antibody complex fluorescent microsphere compound, a preparation method thereof and an NGAL detection kit. The anti-NGAL monoclonal antibody complex fluorescent microsphere compound is obtained by loading an anti-NGAL monoclonal antibody on a complex fluorescent microsphere, the particle size of the complex fluorescent microsphere is 200 nm, the complex fluorescent microsphere is prepared from carbon quantum dots and rare earth europium fluorescent microspheres, the weight ratio of the carbon quantum dots and the rare earth europium fluorescent microspheres is (0.4-0.8):1, and the particle size ratio of the carbon quantum dots and the rare earth europium fluorescent microspheres is (0.04-0.08):1. The anti-NGAL monoclonal antibody complex fluorescent microsphere compound can be used for preparing the NGAL detection kit, and has the advantages of high sensitivity and good stability.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of in vitro reagent detection, more particularly, it relates to an anti-NGAL monoclonal antibody complex fluorescent microsphere complex, a preparation method thereof and an NGAL detection kit. BACKGROUND

[0002] Neutrophil gelatinase-associated lipocalin (NGAL), also known as human lipocalin 2 (Ln2) or siderocalin, is a new member of the human lipocalin family. Because NGAL can be involved in different physiological and pathological processes, NGAL is a marker of acute kidney injury. When early diagnosis of acute kidney injury (AKI) is performed, the concentration of NGAL in blood and urine usually increases rapidly, and the increase is most obvious in 2h (several tens to several hundred times higher than the critical value), while the traditional indicators such as serum creatinine (sCr) and urine enzyme usually increase significantly after 24-72h. Therefore, NGAL can be used for early diagnosis of AKI.

[0003] Currently, the detection methods of NGAL mainly include enzyme-linked immunoassay, radioimmunoassay, Western-blotting, latex-enhanced immunoturbidimetry and chemiluminescence method. Among them, because the latex-enhanced immunoturbidimetry has no high requirement for instruments and equipment, and has no environmental protection and self-protection of operators, compared with other detection methods, this method is simple, rapid, sensitive and reliable, and ordinary automatic or semi-automatic biochemical analyzers can be used, which has a larger application range and greater practical value. For example, the Chinese invention with the publication number CN102590524B discloses a neutrophil gelatinase-associated lipocalin detection kit, the minimum detection limit of which for NGAL antigen is 10.39ng / ml, the linear range is 0-7000ng / ml, and R=0.9982.

[0004] However, the concentration of NGAL in normal urine is lower than 10ng / ml, and such a low concentration puts higher requirements on the sensitivity of the NGAL detection kit. Therefore, the above-mentioned neutrophil gelatinase-associated lipocalin detection kit obviously cannot meet the detection requirements of sensitivity. Therefore, there is an urgent need for a NGAL detection kit with higher sensitivity. SUMMARY

[0005] In order to improve the sensitivity of NGAL detection, the present application provides an anti-NGAL monoclonal antibody complex fluorescent microsphere complex, a preparation method thereof and an NGAL detection kit.

[0006] In the first aspect, the present application provides an anti-NGAL monoclonal antibody complex fluorescent microsphere complex, which adopts the following technical solution:

[0007] An anti-NGAL monoclonal antibody complex fluorescent microsphere complex is obtained by loading an anti-NGAL monoclonal antibody onto a complex fluorescent microsphere, wherein the complex fluorescent microsphere has a particle size of 200 nm;

[0008] The complex fluorescent microsphere is prepared from carbon quantum dots and rare earth europium fluorescent microspheres, and the carbon quantum dots and the rare earth europium fluorescent microspheres

[0009] have a weight ratio of (0.4-0.8):1;

[0010] The carbon quantum dots and the rare earth europium fluorescent microspheres have a particle size ratio of (0.04-0.08):1.

[0011] Rare earth europium is a lanthanide, which has the characteristics of long fluorescence lifetime, narrow emission spectrum, large stokes shift, less background interference, and small biological toxicity. Carbon quantum dots are a new type of fluorescent nanomaterial, which has the advantages of good water solubility, low toxicity, good biological compatibility, and good light stability.

[0012] By using the above technical solution, the carbon quantum dots and the rare earth europium fluorescent microspheres are compounded by promoting the coordination of the carbon quantum dots and the rare earth europium, and are inoculated on the rare earth europium fluorescent microspheres to obtain the complex fluorescent microspheres, which have stronger fluorescence intensity. Because the carbon quantum dots and the rare earth europium fluorescent microspheres have better grading compounding effect at a specific particle size, the structure of the obtained complex fluorescent microspheres is more stable and uniform, which can promote the energy transfer effect of the carbon quantum dots and the rare earth europium in the rare earth europium fluorescent microspheres. In combination with the specific weight ratio of the carbon quantum dots and the rare earth europium fluorescent microspheres, the complex fluorescent microspheres with stronger fluorescence intensity can be prepared with less rare earth europium fluorescent microspheres. Therefore, the carbon quantum dots and the rare earth europium fluorescent microspheres have stronger energy transfer effect at a specific weight and particle size, which promotes the carbon quantum dots to transfer energy to the rare earth europium, thereby improving the fluorescence intensity of the rare earth europium.

[0013] Therefore, the anti-NGAL monoclonal antibody complex fluorescent microsphere complex formed by loading the anti-NGAL monoclonal antibody onto the surface of the complex fluorescent microspheres has stronger fluorescence intensity. When the anti-NGAL monoclonal antibody complex fluorescent microsphere complex is applied to an NGAL detection kit, the NGAL in the sample binds to the anti-NGAL monoclonal antibody complex fluorescent microsphere complex to form an NGAL-anti-NGAL monoclonal antibody complex fluorescent microsphere complex, which is captured by another anti-NGAL monoclonal antibody. Because the fluorescence intensity of the anti-NGAL monoclonal antibody complex fluorescent microsphere complex is strong, the fluorescence analyzer can detect strong fluorescence intensity, thereby improving the sensitivity of the NGAL detection kit in detecting NGAL.

[0014] At the same time, the particle size of the obtained composite fluorescent microspheres is 200 nm, the composite fluorescent microspheres have good specific surface area and more effective groups, the loading capacity of the composite fluorescent microspheres to the anti-NGAL monoclonal antibody is more, and the sensitivity of the final obtained NGAL detection kit to NGAL can be further improved.

[0015] In particular, the rare earth europium itself has certain biological toxicity, in the preparation process of the composite fluorescent microspheres, the addition of the carbon quantum dots reduces the use amount of the rare earth europium, and improves the safety performance of the obtained composite fluorescent microspheres and the anti-NGAL monoclonal antibody composite fluorescent microsphere complex and the NGAL kit.

[0016] Preferably, the weight ratio of the carbon quantum dots to the rare earth europium fluorescent microspheres is (0.4-0.6):1.

[0017] Preferably, the particle size ratio of the carbon quantum dots to the rare earth europium fluorescent microspheres is (0.05-0.08):1.

[0018] By adopting the above technical scheme, by further optimizing the particle size and weight of the carbon quantum dots and the rare earth europium fluorescent microspheres, not only the fluorescence intensity of the obtained composite fluorescent microspheres is improved, but also the fluorescence lifetime of the composite fluorescent microspheres is prolonged. After the composite fluorescent microspheres are loaded with the anti-NGAL monoclonal antibody and applied to the NGAL detection kit, the sensitivity thereof can be improved to 0.2 ng / mL.

[0019] 0.2 ng / mL.

[0020] Preferably, the preparation method of the composite fluorescent microspheres is as follows:

[0021] A1: dissolving the carbon quantum dots in water to obtain a carbon quantum dot aqueous solution; dissolving the rare earth europium fluorescent microspheres in water to obtain a rare earth europium fluorescent microsphere aqueous solution; adding the carbon quantum dot aqueous solution into the rare earth europium fluorescent microsphere aqueous solution, stirring and mixing to obtain a mixed solution;

[0022] A2: reacting the mixed solution at 180-200 DEG C for 3-4 h, naturally cooling to 25-30 DEG C, and performing dialysis purification using a dialysis membrane with a molecular weight cut-off of 1000

[0023] for 20-22 h, and then obtaining the composite fluorescent microspheres by a freeze-drying method.

[0024] By adopting the technical scheme, the carbon quantum dots under the preparation condition are compatible with the rare earth europium fluorescent microspheres, the surface of the carbon quantum dots is activated, the rare earth europium is coordinated on the surface of the carbon quantum dots, and the carbon dot-europium composite fluorescent microspheres are formed. Meanwhile, because the carbon quantum dots have good light stability and the ability to resist external environmental influences, after the carbon quantum dots are coordinated on the surface of the rare earth europium, the light stability and the ability to resist external environmental influences of the obtained composite fluorescent microspheres are improved, the stability of the obtained anti-NGAL monoclonal antibody composite fluorescent microsphere complex is improved, the energy transfer effect between the carbon quantum dots and the rare earth europium is accelerated, the fluorescence intensity of the obtained composite fluorescent microspheres is improved, and the fluorescence intensity of the anti-NGAL monoclonal antibody composite fluorescent microsphere complex is improved. Further, the composite fluorescent microspheres prepared by the method have a large specific surface area and many effective groups, so the loading capacity of the coupled anti-NGAL monoclonal antibody is large. Therefore, the obtained anti-NGAL monoclonal antibody composite fluorescent microsphere complex has high sensitivity and stability when used for preparing an NGAL detection kit.

[0025] Preferably, the rare earth europium fluorescent microspheres are carboxyl-modified rare earth europium fluorescent microspheres.

[0026] By adopting the technical scheme, the carboxyl-modified rare earth europium fluorescent microspheres have a large number of carboxyl groups on the surface, which helps the coordination between the rare earth europium and the carbon quantum dots, and improves the fluorescence intensity and the stability of the formed carbon dot-europium composite fluorescent microspheres.

[0027] In a second aspect, the application provides a preparation method of an anti-NGAL monoclonal antibody composite fluorescent microsphere complex, which adopts the following technical scheme:

[0028] The preparation method of the anti-NGAL monoclonal antibody composite fluorescent microsphere complex comprises the following steps:

[0029] S1: The composite fluorescent microspheres are added into an activation buffer solution with a pH value of 5.8-7.2, and after stirring and mixing, the composite fluorescent microspheres are centrifuged for 3-5 times, and the supernatant is removed to obtain washed composite fluorescent microspheres;

[0030] S2:

[0031] S21: The washed composite fluorescent microspheres are added into a coupling buffer solution with a pH value of 7.2-8.5, and after ultrasonic treatment at 50-65 KHz for 30-40 min, an ultrasonic-treated composite fluorescent microsphere suspension is obtained;

[0032] S22: Add 0.2–1.0 mg / ml of NHS and 0.04–0.2 mg / ml of EDC to the composite fluorescent microsphere suspension, stir and mix, centrifuge 3–5 times, remove the supernatant, and obtain the lower layer of composite fluorescent microspheres.

[0033] Combined fluorescent microsphere stock solution;

[0034] S23: Add the composite fluorescent microsphere stock solution to a coupling buffer with a pH of 7.2–8.5, sonicate at 50–65 kHz for 30–40 min, add anti-NGAL monoclonal antibody, stir and mix, centrifuge 3–5 times, remove the supernatant, and obtain the coupled anti-NGAL monoclonal antibody composite fluorescent microspheres.

[0035] S3: Add the coupled anti-NGAL monoclonal antibody composite fluorescent microspheres to the blocking buffer and sonicate at 50–65 kHz.

[0036] After 30–40 min, blocked anti-NGAL monoclonal antibody composite fluorescent microspheres were obtained;

[0037] The amount of the anti-NGAL monoclonal antibody added is 1-5 mg / mL of the composite fluorescent microsphere stock solution; the activation buffer is any one of PB, Borax, and MES;

[0038] The coupling buffer is any one of PB, Borax, and MES.

[0039] The blocking buffer is one or more of BSA, glycine, casein, fllish skin gelate, Jeffamine D2000, and PEG2000.

[0040] Furthermore, in this application, only PB is used as the activating buffer and coupling buffer, and BSA is used as the blocking buffer, but this does not affect the application of other types of activating buffers, coupling buffers and blocking buffers in this application.

[0041] By using the above technical scheme, the anti-NGAL monoclonal antibody can be loaded on the composite fluorescent microspheres by using EDC and NHS as coupling agents at the same time, so as to improve the stability of the structure of the obtained anti-NGAL monoclonal antibody composite fluorescent microsphere complex. In the process of loading the anti-NGAL monoclonal antibody on the composite fluorescent microspheres, EDC forms an O-isourea structure with the carboxyl group on the anti-NGAL monoclonal antibody composite fluorescent microsphere complex. This activated intermediate product forms an amide cross-linking with the -NH2 on the anti-NGAL monoclonal antibody, and then NHS is used to form a more stable ester, so as to enhance the stability of the EDC cross-linking product. At the same time, EDC and NHS do not actually become part of the anti-NGAL monoclonal antibody composite fluorescent microsphere complex, but are converted into water-soluble urea derivatives, which can be eliminated and washed away, and have no effect on the obtained anti-NGAL monoclonal antibody composite fluorescent microsphere complex. Therefore, when the obtained anti-NGAL monoclonal antibody composite fluorescent microsphere complex is used to prepare an NGAL detection test kit, the sensitivity of the obtained NGAL detection test kit can be improved.

[0042] Preferably, the anti-NGAL monoclonal antibody is added in an amount of 3 mg / mL of the composite fluorescent microsphere stock solution.

[0043] By using the above technical scheme, under the above amount of anti-NGAL monoclonal antibody, the loading amount of the anti-NGAL monoclonal antibody coupled to the composite fluorescent microspheres is the highest, the obtained anti-NGAL monoclonal antibody composite fluorescent microsphere complex has a certain weight, the loss of the anti-NGAL monoclonal antibody composite fluorescent microsphere complex during preparation can be reduced, and the stability and sensitivity of the structure of the obtained anti-NGAL monoclonal antibody composite fluorescent microsphere complex can be improved.

[0044] In a third aspect, the present application provides an NGAL detection test kit, which adopts the following technical scheme:

[0045] An NGAL detection test kit includes an NGAL detection test card coated with an anti-NGAL monoclonal antibody composite fluorescent microsphere complex.

[0046] Preferably, the minimum detection limit is 0.5 ng / mL, the linear range is 0-500 ng / mL, and the correlation coefficient R2 is greater than 0.99.

[0047] By using the above technical scheme, the obtained NGAL detection test kit has a minimum detection limit of NGAL as low as

[0048] It has a concentration of 0.5 ng / mL and exhibits good linearity within the NGAL concentration range of 0–500 ng / mL, with a correlation coefficient R0. 2 Gundam

[0049] The value is above 0.99, which indicates that the NGAL detection kit obtained in this application has a high sensitivity for NGAL detection.

[0050] In summary, this application has the following beneficial effects:

[0051] 1. Because this application uses carbon quantum dots of specific weight and particle size to compound with rare earth europium fluorescent microspheres, by promoting the coordination of carbon quantum dots with rare earth europium, the resulting composite fluorescent microspheres not only have a larger specific surface area, which can increase the content of anti-NGAL monoclonal antibody, but also promote the energy transfer effect between carbon quantum dots and rare earth europium in the rare earth europium fluorescent microspheres, accelerating the transfer of energy from carbon quantum dots to rare earth europium, and increasing the fluorescence intensity of the resulting composite fluorescent microspheres. After applying the composite fluorescent microspheres to the NGAL detection kit, the detection sensitivity of the NGAL detection kit can be improved.

[0052] 2. In this application, by further optimizing the particle size and weight of carbon quantum dots and rare earth europium fluorescent microspheres, the energy transfer effect of rare earth europium in carbon quantum dots and rare earth europium fluorescent microspheres can be further promoted. After loading the obtained composite fluorescent microspheres with anti-NGAL monoclonal antibody, it can be applied to the NGAL detection kit, and its sensitivity can be improved to 0.2 ng / mL.

[0053] 3. In the process of preparing composite fluorescent microspheres, carbon quantum dots and rare earth europium fluorescent microspheres have good compatibility, which promotes the activation of the carbon quantum dot surface. This promotes the coordination of rare earth europium on the carbon quantum dot surface to form carbon dot-europium composite fluorescent microspheres. After loading the obtained composite fluorescent microspheres with anti-NGAL monoclonal antibody, they can be used in NGAL detection kits to improve their sensitivity and stability. Attached Figure Description

[0054] Figure 1 is a schematic diagram of the overall structure of the NGAL detection kit according to an embodiment of this application;

[0055] Figure 2 is a standard curve of fluorescence intensity ratio T / C versus antigen concentration in Application Example 1 of this application;

[0056] Figure 3 is a standard curve of fluorescence intensity ratio T / C versus antigen concentration in Application Example 2 of this application;

[0057] Figure 4 is a standard curve of fluorescence intensity ratio T / C versus antigen concentration in Application Example 3 of this application;

[0058] Figure 5 is a standard curve of the fluorescence intensity ratio T / C versus the concentration of the antigen in the application Example 4;

[0059] Figure 6 is a standard curve of the fluorescence intensity ratio T / C versus the concentration of the antigen in the application Example 5;

[0060] Figure 7 is a standard curve of the fluorescence intensity ratio T / C versus the concentration of the antigen in the application Example 6;

[0061] Figure 8 is a standard curve of the fluorescence intensity ratio T / C versus the concentration of the antigen in the application Example 7;

[0062] Figure 9 is a standard curve of the fluorescence intensity ratio T / C versus the concentration of the antigen in the application Example 8;

[0063] Figure 10 is a standard curve of the fluorescence intensity ratio T / C versus the concentration of the antigen in the application Example 9;

[0064] Figure 11 is a standard curve of the fluorescence intensity ratio T / C versus the concentration of the antigen in the application Example 10;

[0065] Figure 12 is a standard curve of the fluorescence intensity ratio T / C versus the concentration of the antigen in the application Example 11;

[0066] Figure 13 is a standard curve of the fluorescence intensity ratio T / C versus the concentration of the antigen in the application Comparative Example 1;

[0067] Figure 14 is a standard curve of the fluorescence intensity ratio T / C versus the concentration of the antigen in the application Comparative Example 2;

[0068] Figure 15 is a standard curve of the fluorescence intensity ratio T / C versus the concentration of the antigen in the application Comparative Example 3;

[0069] Figure 16 is a standard curve of the fluorescence intensity ratio T / C versus the concentration of the antigen in the application Comparative Example 4;

[0070] Figure 17 is a standard curve of the fluorescence intensity ratio T / C versus the concentration of the antigen in the application Comparative Example 5.

[0071] Reference signs: 1, PVC base plate; 2, binding pad; 3, detection line; 4, quality control line. DETAILED DESCRIPTION

[0072] The application will be further described in detail below with reference to the accompanying drawings and examples.

[0073] The raw materials used in the embodiments of the present application are commercially available, except for the special description below: The anti-NGAL monoclonal antibody with a model number of FAP-KC001 and a purity of greater than or equal to 95% is purchased from Suncroft;

[0074] The rare earth europium fluorescent microspheres are rare earth europium fluorescent carboxyl microspheres with a model number of BM100-010-EU and are purchased from Shanghai Yutao International Trade Co., Ltd.;

[0075] The rare earth europium fluorescent microspheres are carboxyl and sulfonic acid group modified rare earth europium fluorescent microspheres with a particle size of 100-200 nm and are purchased from Shanghai Yutao International Trade Co., Ltd.

[0076] The carbon quantum dots have a model number of QDC620 and a particle size of 3-9 nm and are purchased from KunDao Biotech Co., Ltd.; The goat anti-mouse IgG has a model number of FAB-S003 and is purchased from Suncroft.

[0077] The freeze dryer has a model number of LGI-10N and is purchased from Yaxing Science and Technology Co., Ltd.

[0078] Preparation examples of raw materials

[0079] Preparation Example 1

[0080] A composite fluorescent microsphere is prepared by the following steps:

[0081] A1: 1 g of rare earth europium fluorescent microspheres and 0.4 g of carbon quantum dots are sequentially added to 14 g of deionized water, and stirred and mixed at 25°C, 3000 r / min

[0082] for 10 min to obtain a mixed solution;

[0083] A2: The mixed solution is transferred to a reaction kettle, and after reaction at 170°C for 2.5 h, it is naturally cooled to 25°C, and then purified by dialysis for 19 h using a dialysis membrane with a molecular weight cut-off of 1000. The purified mixed solution is freeze-dried by a freeze dryer for 24 h to obtain the composite fluorescent microsphere.

[0084] The particle size of the carbon quantum dots is 4 nm.

[0085] The rare earth europium fluorescent microspheres are carboxyl and sulfonic acid group modified rare earth europium fluorescent microspheres with a particle size of 100 nm; The particle size of the obtained composite fluorescent microsphere is 200 nm.

[0086] Preparation Example 2

[0087] A composite fluorescent microsphere is prepared by the following steps:

[0088] A1: 0.4g carbon quantum dots were dissolved in 4g deionized water to obtain a carbon quantum dot aqueous solution; 1g rare earth europium fluorescent microspheres were dissolved in 10g deionized water to obtain a rare earth europium fluorescent microsphere aqueous solution; the carbon quantum dot aqueous solution was added to the rare earth europium fluorescent microsphere aqueous solution, and the mixture was stirred at 25°C and 3000r / min for 10min to obtain a mixed solution;

[0089] A2: The mixed solution was transferred to a reaction kettle, and reacted at 180-200°C (180°C in this example) for 3-4h (3.5h in this example), and then naturally cooled to 25°C. Then, the purified mixed solution was dialyzed for 20-22h (21h in this example) using a dialysis membrane with a molecular weight cut-off of 1000, and then freeze-dried for 24h to obtain the composite fluorescent microspheres.

[0090]

[0091] The particle size of the carbon quantum dots was 4nm;

[0092] The rare earth europium fluorescent microspheres were carboxyl and sulfonic acid group modified rare earth europium fluorescent microspheres with a particle size of 100nm; and the particle size of the obtained composite fluorescent microspheres was 200nm.

[0093] Preparation Examples 3-5

[0094] A composite fluorescent microsphere, which was different from Preparation Example 2, was the same as Preparation Example 1 except that the weight of the carbon quantum dots and deionized water was different in the preparation step A1. The weight of the carbon quantum dots was shown in the following table.

[0095]

[0096] Preparation Examples 6-8

[0097] A composite fluorescent microsphere, which was different from Preparation Example 3, was the same as Preparation Example 3 except that the particle size of the carbon quantum dots was different. The particle size of the carbon quantum dots was shown in the following table.

[0098]

[0099] Preparation Example 9

[0100] A composite fluorescent microsphere, which was different from Preparation Example 7, was the same as Preparation Example 1 except that the rare earth europium fluorescent microspheres were carboxyl modified rare earth europium fluorescent microspheres.

[0101] Example Example 1

[0102] ​A kind of anti-NGAL monoclonal antibody complex fluorescent microsphere complex, by anti-NGAL monoclonal antibody is loaded on complex fluorescent microsphere, its preparation steps are as follows:

[0103] S1:1g complex fluorescent microsphere is added to 9mL, pH value is 5.8~7.2 (pH value is 6.5 in this embodiment) activation buffer solution, under the condition of 25 ℃, 2000r / min stirring mixing 1h, then centrifugation 4 times at 3000r / min, every time 1min, remove supernatant, to obtain the washed complex fluorescent microsphere.

[0104] S2:the washed complex fluorescent microsphere obtained in S1 is added to 30mL, pH value is 7.2~8.5 (pH value is 7.9 in this embodiment) coupling buffer solution, after 50 KHz ultrasonic 30 min, obtain the ultrasonic complex fluorescent microsphere suspension;

[0105] 0.2 ~ 1.0mg / ml complex fluorescent microsphere suspension of NHS (0.6mg / ml complex fluorescent microsphere suspension in this embodiment) and 0.04~0.2mg / ml complex fluorescent microsphere suspension of EDC (0.1mg / ml complex fluorescent microsphere suspension in this embodiment) are added to complex fluorescent microsphere suspension, under the condition of 25 ℃, 2000r / min stirring mixing 1h, then centrifugation 4 times at 15000r / min, every time 1min, remove supernatant, to obtain the lower complex fluorescent microsphere stock solution;

[0106] The complex fluorescent microsphere stock solution is added to 30mL, pH value is 7.2~8.5 (pH value is 7.9 in this embodiment) coupling buffer solution, after 50~65 KHz (50 KHz in this embodiment) ultrasonic 30~40 min (30min in this embodiment), 1mg / mL complex fluorescent microsphere stock solution anti-NGAL monoclonal antibody is added, under the condition of 25 ℃, 2000r / min stirring mixing

[0107] 3h, then centrifugation 4 times at 15000r / min, every time 1min, remove supernatant, to obtain the coupled anti-NGAL monoclonal antibody complex fluorescent microsphere;

[0108] S3: The coupled anti-NGAL monoclonal antibody complex fluorescent microspheres are added into the blocking buffer, and after ultrasonic treatment at 50-65 KHz (50 KHz in this embodiment) for 30-40 min (30 min in this embodiment), the blocked anti-NGAL monoclonal antibody complex fluorescent microspheres are obtained.

[0109] The activation buffer is PB, and the coupling buffer is PB.

[0110] The blocking buffer is BSA.

[0111] The complex fluorescent microspheres are prepared according to Preparation Example 1.

[0112] Embodiments 2-9

[0113] An anti-NGAL monoclonal antibody complex fluorescent microsphere complex is different from that of Embodiment 1 in that the complex fluorescent microspheres obtained according to Preparation Examples 2-9 are used, and other aspects are the same as those of Embodiment 1.

[0114] Embodiments 10-11

[0115] An anti-NGAL monoclonal antibody complex fluorescent microsphere complex is different from that of Embodiment 9 in that the amount of anti-NGAL monoclonal antibody added is different, and other aspects are the same as those of Embodiment 9. The amount of anti-NGAL monoclonal antibody added is shown in the following table.

[0116]

[0117] Application Embodiments

[0118] Application Embodiments 1-11

[0119] An NGAL detection kit includes an NGAL detection test card, a sample diluent, and an IC card.

[0120] Referring to FIG. 1, the NGAL detection test card includes a PVC bottom plate 1 and a sample pad, a conjugate pad 2, an NC membrane, and an absorbent pad arranged in sequence on the PVC bottom plate, and the NC membrane has a detection line (T line) 3 and a quality control line (C line) 4. The T line is coated with anti-NGAL monoclonal antibody, and the C line is coated with sheep anti-mouse IgG. The conjugate pad is coated with the anti-NGAL monoclonal antibody complex fluorescent microsphere complex prepared according to Embodiments 1-11.

[0121] The sample diluent is a 0.1M PB buffer; and the IC card has copied therein information such as relevant parameters and operation steps of the detection process using the kit.

[0122] The instrument used in the present test kit is AFS3000B dry fluorescence immunoassay analyzer produced by Guangzhou Lanbo Biological Technology Co., Ltd.

[0123] Sensitivity detection

[0124] Preparation of standard working solution: NGAL antigen was prepared into a series of concentrations (0; 0.2 ng / mL; 0.5 ng / mL; 1 ng / mL; 5 ng / mL; 10 ng / mL; 50 ng / mL; 100 ng / mL; 500 ng / mL) with 5% calf serum PBS buffer (0.02M, pH 7.4), 80 μL was taken by a pipette and added to the binding pad, after 5 minutes, the fluorescence intensity of T line and C line was detected by AFS3000B dry fluorescence immunoassay analyzer produced by Guangzhou Lanbo Biological Technology Co., Ltd., three times each time, the average value was taken, then the fluorescence intensity ratio T / C of T line and C line was calculated and recorded in the following table.

[0125]

[0126] By analyzing the data in the above table, it can be seen that the sensitivity of the NGAL detection test kit obtained by applying examples 1-5 for detecting NGAL antigen can reach 0.5 ng / mL, and the sensitivity of the NGAL detection test kit obtained by applying examples 6-11 for detecting NGAL antigen can reach 0.2 ng / mL.

[0127] According to the fluorescence intensity ratio T / C and the antigen concentration, a standard curve was drawn, the results are shown in Figures 2-12.

[0128] Referring to Figure 2, in the application example 1, the standard curve equation for detecting NGAL antigen is y=1.0115x+14.001, R²=0.9986, the linear range is 0-500 ng / mL.

[0129] Referring to Figure 3, in the application example 2, the standard curve equation for detecting NGAL antigen is y=1.0311x+14.116, R²=0.9988, the linear range is 0-500 ng / mL.

[0130] Referring to Figure 4, in the application example 3, the standard curve equation for detecting NGAL antigen is y=1.0683x+14.929, R²=0.9987, the linear range is 0-500 ng / mL.

[0131] Referring to FIG. 5, in the application embodiment 4, the standard curve equation for detecting NGAL antigen is y = 1.0954x + 15.611, R2= 0.9986, and the linear range is 0-500 ng / mL.

[0132] Referring to FIG. 6, in the application embodiment 5, the standard curve equation for detecting NGAL antigen is y = 1.1095x + 17.014, R2= 0.9987, and the linear range is 0-500 ng / mL.

[0133] Referring to FIG. 7, in the application embodiment 6, the standard curve equation for detecting NGAL antigen is y = 1.1212x + 18.479, R2= 0.9988, and the linear range is 0-500 ng / mL.

[0134] Referring to FIG. 8, in the application embodiment 7, the standard curve equation for detecting NGAL antigen is y = 1.1138x + 19.547, R2= 0.9989, and the linear range is 0-500 ng / mL.

[0135] Referring to FIG. 9, in the application embodiment 8, the standard curve equation for detecting NGAL antigen is y = 1.1472x + 20.475, R2= 0.9987, and the linear range is 0-500 ng / mL.

[0136] Referring to FIG. 10, in the application embodiment 9, the standard curve equation for detecting NGAL antigen is y = 1.1525x + 22.129, R2= 0.9987, and the linear range is 0-500 ng / mL.

[0137] Referring to FIG. 11, in the application embodiment 10, the standard curve equation for detecting NGAL antigen is y = 1.1731x + 23.217, R2= 0.9987, and the linear range is 0-500 ng / mL.

[0138] Referring to FIG. 12, in the application embodiment 11, the standard curve equation for detecting NGAL antigen is y = 1.1909x + 24.504, R2= 0.9987, and the linear range is 0-500 ng / mL.

[0139] In summary, the NGAL detection kit obtained in the application embodiments 1-11 has high sensitivity for detecting NGAL antigen, and has good linear relationship and correlation coefficient R2in the concentration range of 0-500 ng / mL of NGAL antigen.2 All can reach 0.99 or more.

[0140] Stability test

[0141] The NGAL antigen was prepared into 1 ng / mL, 10 ng / mL and 100 ng / mL test samples with 5% calf serum PBS buffer (0.02M, pH 7.4), 80 μL was taken with a pipette gun and added to the binding pad, after 5 minutes, the fluorescence intensity of the T line was detected with the AFS3000B dry fluorescence immunoassay analyzer produced by Guangzhou Lanbo Biological Technology Co., Ltd., 10 times of detection were performed each time, the average deviation CV% value was calculated according to the 10 times of detection results, and was recorded in the following table.

[0142]

[0143] By analyzing the data in the above table, when the NGAL antigen of 1 ng / mL is used as the test sample, 10 times of continuous detection are performed, and the CV% value of the detection reagent kit of embodiments 1-11 is only 3.98-4.55%; when the NGAL antigen of 10 ng / mL is used as the test sample, 10 times of continuous detection are performed, and the CV% value of the detection reagent kit of embodiments 1-11 is only 3.30-4.13%; when the NGAL antigen of 100 ng / mL is used as the test sample, 10 times of continuous detection are performed, and the CV% value of the detection reagent kit of embodiments 1-11 is only 3.10-4.02%.

[0144] It is thus shown that the NGAL detection reagent kit obtained by embodiments 1-11 has high stability.

[0145] Comparative example

[0146] Comparative example 1

[0147] A kind of anti-NGAL monoclonal antibody fluorescent microsphere complex, which is different from embodiment 1, except that the complex fluorescent microspheres are carboxyl and sulfonic acid group modified rare earth europium fluorescent microspheres, the particle size is 200 nm, the model is EU300-10, and is purchased from Shanghai Yutao International Trade Co., Ltd.

[0148] Comparative example 2

[0149] A kind of anti NGAL monoclonal antibody fluorescent microsphere compound, different from the embodiment 1, except that the weight of carbon quantum dots and rare earth europium fluorescent microspheres is different in the preparation step of compound fluorescent microspheres, other is same with the embodiment 1.The preparation steps of compound fluorescent microspheres are as follows:

[0150] A1: 1g of rare earth europium fluorescent microspheres and 0.3g of carbon quantum dots are sequentially added to 13g of deionized water, stirred and mixed at 25 DEG C and 3000r / min for 10min to obtain a mixed solution;

[0151] A2: the mixed solution is transferred to a reaction kettle, reacted at 170 DEG C for 2.5h, then naturally cooled to 25 DEG C, then purified by dialysis membrane with a molecular weight cut-off of 1000 for 19h, and the purified mixed solution is freeze-dried by freeze-drier for 24h to obtain compound fluorescent microspheres.

[0152] The particle size of the carbon quantum dots is 4nm;The rare earth europium fluorescent microspheres are carboxyl and sulfonic acid group modified rare earth europium fluorescent microspheres, and the particle size is 100nm;The particle size of the obtained compound fluorescent microspheres is 200nm.

[0153] Comparative example 3

[0154] A kind of anti NGAL monoclonal antibody fluorescent microsphere compound, different from the embodiment 1, except that the weight of carbon quantum dots and rare earth europium fluorescent microspheres is different in the preparation step of compound fluorescent microspheres, other is same with the embodiment 1.The preparation steps of compound fluorescent microspheres are as follows:

[0155] A1: 1g of rare earth europium fluorescent microspheres and 0.9g of carbon quantum dots are sequentially added to 19g of deionized water, stirred and mixed at 25 DEG C and 3000r / min for 10min to obtain a mixed solution;

[0156] A2: the mixed solution is transferred to a reaction kettle, reacted at 170 DEG C for 2.5h, then naturally cooled to 25 DEG C, then purified by dialysis membrane with a molecular weight cut-off of 1000 for 19h, and the purified mixed solution is freeze-dried by freeze-drier for 24h to obtain compound fluorescent microspheres.

[0157] The particle size of the carbon quantum dots is 4nm;

[0158] The rare earth europium fluorescent microspheres are carboxyl and sulfonic acid group modified rare earth europium fluorescent microspheres, and the particle size is 100nm;The particle size of the obtained compound fluorescent microspheres is 200nm.

[0159] Comparative example 4

[0160] An anti-NGAL monoclonal antibody fluorescent microsphere complex, which is different from Example 1 in that, in addition to the preparation process of the complex fluorescent microspheres, the particle size of the carbon quantum dots is 3 nm, and the particle size of the rare earth europium fluorescent microspheres is 100 nm, and the others are the same as Example 1.

[0161] Comparative Example 5

[0162] An anti-NGAL monoclonal antibody fluorescent microsphere complex, which is different from Example 1 in that, in addition to the preparation process of the complex fluorescent microspheres, the particle size of the carbon quantum dots is 9 nm, and the particle size of the rare earth europium fluorescent microspheres is 100 nm, and the others are the same as Example 1.

[0163] In the above-mentioned Comparative Examples 2-5, the weight and particle size of the carbon quantum dots and the rare earth europium fluorescent microspheres are shown in the following table.

[0164]

[0165] Application of Comparative Examples 1-5

[0166] An NGAL detection kit, which is different from Application Example 1 in that, in addition to the binding pad being replaced by the anti-NGAL monoclonal antibody fluorescent microsphere complex prepared in Comparative Examples 1-5 instead of the anti-NGAL monoclonal antibody complex fluorescent microsphere complex prepared in Example 1, the others are the same as Application Example 1.

[0167] Sensitivity detection

[0168] Preparation of standard working solution: NGAL antigen was prepared into a series of concentrations (0; 0.2 ng / mL; 0.5 ng / mL; 1 ng / mL; 5 ng / mL; 10 ng / mL; 50 ng / mL; 100 ng / mL; 500 ng / mL) with 5% calf serum PBS buffer (0.02M, pH7.4), 80μL was taken with a pipette and added to the binding pad, after 5 minutes, the fluorescence intensity of T line and C line was detected by AFS3000B dry fluorescence immunoassay analyzer produced by Guangzhou Lanbo Biological Technology Co., Ltd., and the fluorescence intensity ratio T / C of T line and C line was calculated, and the detection results were taken as the average value, and then recorded in the following table.

[0169]

[0170] From the data in the above table, it can be seen that the NGAL detection kit obtained by using the comparative example 1 has a smaller variation range of the fluorescence intensity ratio T / C when the concentration of NGAL antigen is 0-5 ng / mL. Therefore, the minimum detection amount of the NGAL detection kit obtained by using the comparative example 1 is 10 ng / mL, which is much higher than the minimum detection amount 0.5 ng / mL of the NGAL detection kit obtained by using the application example 1. Thus, it is shown that the NGAL detection kit obtained by using the anti-NGAL monoclonal antibody complexed with the fluorescent microspheres obtained by using the application examples 1-11 has a lower minimum detection amount, i.e., a higher sensitivity, as compared with the commercially available NGAL detection kit.

[0171] The NGAL detection kits obtained by using the comparative examples 2-3 have a smaller variation range of the fluorescence intensity ratio T / C when the concentration of NGAL antigen is 0-0.5 ng / mL. Therefore, the minimum detection amount of the NGAL detection kit obtained by using the comparative examples 2-3 is 1 ng / mL, which is much lower than the minimum detection amount 0.5 ng / mL of the NGAL detection kit obtained by using the application example 1. Thus, it is shown that the NGAL detection kit obtained by using the anti-NGAL monoclonal antibody complexed with the fluorescent microspheres obtained by using the application example 1 has a lower minimum detection amount, i.e., a higher sensitivity. The reason is that, in the preparation of the anti-NGAL monoclonal antibody complexed with the fluorescent microspheres, the complexed fluorescent microspheres are prepared by using carbon quantum dots and rare earth europium fluorescent microspheres at a weight ratio of (0.4-0.8):1, and the fluorescence energy transfer from the carbon quantum dots to the europium ions occurs in the obtained complexed fluorescent microspheres, so that the energy of the carbon quantum dots is transferred to the europium ions, resulting in the enhancement of the fluorescence intensity of the europium ions, and thus the sensitivity of the obtained NGAL detection kit for detecting NGAL antigen is improved.

[0172] The fluorescence intensity ratio T / C of the NGAL detection kit obtained from Comparative Examples 4-5 varied slightly when the concentration of NGAL antigen was 0-0.5 ng / mL. Therefore, the minimum detection amount of the NGAL detection kit obtained from Comparative Examples 4-5 was 1 ng / mL, which was much lower than the minimum detection amount of the NGAL detection kit obtained from Example 1. This indicates that the NGAL detection kit obtained from Example 1 has a lower minimum detection amount, i.e., higher sensitivity. The reason for this may be that, during the preparation of the composite fluorescent microspheres, when the particle size ratio of carbon quantum dots to rare earth europium fluorescent microspheres was (0.04-0.08):1, the structure of the obtained composite fluorescent microspheres was more stable, which was conducive to improving the stability of the structure of the antibody cross-linking product of the composite fluorescent microspheres, so that the NGAL detection kit obtained from Example 1 has higher sensitivity and higher stability when detecting NGAL antigen.

[0173] The standard curve of fluorescence intensity ratio T / C versus antigen concentration was plotted, and the results are shown in FIGS. 2-12.

[0174] Referring to FIG. 13, the standard curve equation for detecting NGAL antigen in Comparative Example 1 was y=0.9586x+12.329, R²=0.9985, and the linear range was 0-500 ng / mL.

[0175] Referring to FIG. 14, the standard curve equation for detecting NGAL antigen in Comparative Example 2 was y=0.953x+1.713, R²=0.9989, and the linear range was 0-500 ng / mL.

[0176] Referring to FIG. 15, the standard curve equation for detecting NGAL antigen in Comparative Example 3 was y=0.9492x+14.549, R²=0.9982, and the linear range was 0-500 ng / mL.

[0177] Referring to FIG. 16, the standard curve equation for detecting NGAL antigen in Comparative Example 4 was y=0.9601x+14.123, R²=0.9981, and the linear range was 0-500 ng / mL.

[0178] Referring to FIG. 17, the standard curve equation for detecting the NGAL antigen in Comparative Example 5 is y = 0.9658x + 13.962, R2= 0.9988, and the linear range is 0-500 ng / mL.

[0179] Stability test

[0180] The NGAL antigen was prepared into three concentrations of 50 ng / mL, 100 ng / mL and 500 ng / mL of the test sample using 5% calf serum PBS buffer (0.02M, pH 7.4), 80 μL was taken by a pipette and added to the binding pad, after 5 minutes, the fluorescence intensity of the T line was detected by the AFS3000B dry fluorescence immunoassay analyzer produced by Guangzhou Lanbo Biological Technology Co., Ltd., 10 times of detection were performed each time, the average deviation CV% value was calculated according to the 10 times of detection results, and was recorded in the following table.

[0181]

[0182] It can be seen from the analysis of the above table data that when the NGAL antigen with concentrations of 50 ng / mL, 100 ng / mL and 500 ng / mL is used as the test sample, 10 times of continuous detection are performed, and the CV% value of the detection results of the kit of Comparative Example 1 is much larger than the CV% value of the kit of Example 1. Therefore, it is shown that the anti-NGAL monoclonal antibody complex fluorescent microsphere complex obtained by Examples 1-11 and the NGAL detection kit finally obtained have good stability relative to the commercially available NGAL detection kit.

[0183] It can be seen from the analysis of the above table data that when the NGAL antigen with concentrations of 50 ng / mL, 100 ng / mL and 500 ng / mL is used as the test sample, 10 times of continuous detection are performed, and the CV% value of the detection results of the kit of Comparative Example 2-3 is much larger than the CV% value of the kit of Example 1. Therefore, it is shown that in the preparation process of the anti-NGAL monoclonal antibody complex fluorescent microsphere complex, when the complex fluorescent microspheres are prepared by carbon quantum dots and rare earth europium fluorescent microspheres in a weight ratio of (0.4-0.8):1, the anti-NGAL monoclonal antibody complex fluorescent microsphere complex obtained has high stability for preparing the NGAL detection kit.

[0184] By analyzing the data in the above table, it can be seen that when the NGAL antigen with three concentrations of 50 ng / mL, 100 ng / mL and 500 ng / mL is used as the test sample, the CV% value of the detection results of the detection reagent kit of Comparative Examples 5-5 is much larger than the CV% value of the application of Example 1. It is shown that in the preparation process of the anti-NGAL monoclonal antibody complex fluorescent microsphere complex, when the particle size ratio of the complex fluorescent microspheres prepared from carbon quantum dots and rare earth europium fluorescent microspheres is (0.04-0.08):1, the obtained anti-NGAL monoclonal antibody complex fluorescent microsphere complex is used for preparing the NGAL detection reagent kit, and the obtained NGAL detection reagent kit has high stability.

[0185] The specific embodiments are only an explanation of the present application, which is not a limitation of the present application. Those skilled in the art can make modifications to the embodiments without creative contribution after reading the specification, but as long as it is within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. A complex of anti-NGAL monoclonal antibody and fluorescent microspheres, characterized in that, It was obtained by loading anti-NGAL monoclonal antibody onto composite fluorescent microspheres, wherein the particle size of the composite fluorescent microspheres is 200 nm; The composite fluorescent microspheres are prepared from carbon quantum dots and rare earth europium fluorescent microspheres, with a weight ratio of carbon quantum dots to rare earth europium fluorescent microspheres of (0.4-0.8):

1. The particle size ratio of carbon quantum dots to rare earth europium fluorescent microspheres is (0.04–0.08):1; The method for preparing the composite fluorescent microspheres is as follows: A1: Dissolve carbon quantum dots in water to obtain an aqueous solution of carbon quantum dots; dissolve rare earth europium fluorescent microspheres in water to obtain an aqueous solution of rare earth europium fluorescent microspheres; add the aqueous solution of carbon quantum dots to the aqueous solution of rare earth europium fluorescent microspheres, stir and mix to obtain a mixed solution; A2: The mixed solution was reacted at 180-200℃ for 3-4 hours, then naturally cooled to 25-30℃. After dialysis purification using a dialysis membrane with a molecular weight cutoff of 1000 for 20-22 hours, the composite fluorescent microspheres were obtained by lyophilization. The rare earth europium fluorescent microspheres are carboxyl-modified rare earth europium fluorescent microspheres.

2. The anti-NGAL monoclonal antibody-fluorescent microsphere complex according to claim 1, characterized in that, The weight ratio of the carbon quantum dots to the rare earth europium fluorescent microspheres is (0.4–0.6):

1.

3. The anti-NGAL monoclonal antibody-fluorescent microsphere complex according to claim 1, characterized in that, The particle size ratio of the carbon quantum dots to the rare earth europium fluorescent microspheres is (0.05–0.08):

1.

4. The method for preparing the anti-NGAL monoclonal antibody complex with fluorescent microspheres according to any one of claims 1 to 3, characterized in that, Includes the following steps: S1: Add the composite fluorescent microspheres to an activation buffer solution with a pH of 5.8–7.2, stir and mix, centrifuge 3–5 times, remove the supernatant, and obtain the washed composite fluorescent microspheres; S2： S21: The cleaned composite fluorescent microspheres were added to a coupling buffer solution with a pH of 7.2–8.5 and sonicated at 50–65 kHz for 30–40 min to obtain a sonicated suspension of composite fluorescent microspheres. S22: Add NHS with a final concentration of 0.2-1.0 mg / ml and EDC with a final concentration of 0.04-0.2 mg / ml to the composite fluorescent microsphere suspension, stir and mix, centrifuge 3-5 times, remove the supernatant, and obtain the lower layer of composite fluorescent microsphere stock solution; S23: Add the composite fluorescent microsphere stock solution to a coupling buffer with a pH of 7.2-8.5, sonicate at 50-65 kHz for 30-40 min, add anti-NGAL monoclonal antibody at a final concentration of 1-5 mg / mL, stir and mix, centrifuge 3-5 times, remove the supernatant, and obtain the coupled anti-NGAL monoclonal antibody composite fluorescent microspheres. S3 blocking: The coupled anti-NGAL monoclonal antibody composite fluorescent microspheres were added to the blocking buffer and sonicated at 50-65 kHz for 30-40 min to obtain blocked anti-NGAL monoclonal antibody composite fluorescent microspheres. The activation buffer is any one of PB, Borax and MES; The coupling buffer is any one of PB, Borax, and MES; The blocking buffer is one or more of BSA, glycine, casein, fllish skin gelate, Jeffamine D2000, and PEG2000.

5. The method for preparing the anti-NGAL monoclonal antibody-fluorescent microsphere complex according to claim 4, characterized in that, In step S23, an anti-NGAL monoclonal antibody with a final concentration of 3 mg / mL is added.

6. An NGAL detection kit, characterized in that, The NGAL detection reagent card includes an anti-NGAL monoclonal antibody complex fluorescent microsphere complex as described in any one of claims 1 to 3.

7. The NGAL detection kit according to claim 6, characterized in that, The kit has a limit of detection of 0.5 ng / mL, a linear range of 0–500 ng / mL, and a correlation coefficient R0. 2 >0.99.

Images