Fluorescent reagent for human neutrophil apolipoprotein detection and application thereof

Abstract

The invention relates to a fluorescent reagent for detecting human neutrophil apolipoprotein and application of the fluorescent reagent. The fluorescent reagent comprises a fluorescent reagent I and a fluorescent reagent II, the fluorescent reagent I and the fluorescent reagent II are encapsulated in polymethyl methacrylate microspheres and have single-excitation dual-emission characteristics, the excitation light is near 345 nm, the emission light is near 445 nm and 615 nm respectively, and the emission light of 615 nm is later than the emission light of 445 nm. The reagent, a buffer solution and human neutrophil apolipoprotein antibody protein form a detection kit, and the detection kit is used for detecting neutrophil apolipoprotein in fresh human venous blood. The preparation method comprises the steps of fluorescent microsphere preparation, antibody preparation, microsphere surface activation, coupling reaction, sealing, cleaning, storage and the like. The detection method comprises the following steps: taking serum, mixing the fluorescent microsphere compound, detecting a fluorescence value and calculating the protein concentration. The method improves the sensitivity and accuracy of a detection system, is simple and convenient to operate and short in time consumption, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the fields of medical biology, etc., and specifically relates to a fluorescent reagent for detecting human neutrophil apolipoprotein and its application. Background Art

[0002] Human neutrophil apolipoprotein is a protein mainly secreted by human neutrophils. It has the characteristics of fast inflammatory mediation reaction speed and strong signal specificity, and is suitable for clinical detection to characterize the occurrence and development degree of inflammation. Currently, the existing detection methods are as follows: 1. Immunoturbidimetry. This is a commonly used detection method. Based on the specific binding of antigen and antibody to form an immune complex, which causes a change in the turbidity of the solution, and the content of neutrophil apolipoprotein is quantitatively detected by measuring the change in turbidity with a turbidimeter. This method is simple and fast to operate, but has poor specificity and accuracy. 2. Enzyme-linked immunosorbent assay (ELISA): ELISA is a highly sensitive and specific detection method. The antigen or antibody is coated on the surface of a solid-phase carrier, the sample to be detected and the enzyme-labeled antibody or antigen are added, and the content of neutrophil apolipoprotein is quantitatively detected by the enzyme-catalyzed substrate color development. The ELISA method has high sensitivity and specificity, but the operation is relatively complex and time-consuming. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a fluorescent reagent for detecting human neutrophil apolipoprotein and its application to solve the deficiencies mentioned in the prior art.

[0004] The technical solution of the present invention is as follows: A fluorescent reagent for detecting human neutrophil apolipoprotein, including fluorescent reagent I and fluorescent reagent II, with the structural formulas as follows:

[0005] Fluorescent reagent I and fluorescent reagent II are encapsulated together in microspheres based on polymethyl methacrylate in the liquid phase to form fluorescent microspheres. The two fluorescent substances do not interfere with each other, and their excitation lights are both around 345 nm, and the emission lights are respectively around 445 nm and 615 nm. Among them, the emission light at 615 nm is later than the emission light at 445 nm, and the interval is at the microsecond level. This design enables the two fluorescent substances to complement each other's advantages and improves the sensitivity and accuracy of the detection system.

[0006] Furthermore, the present invention also provides the application of the above fluorescent reagent in a kit for detecting human neutrophil apolipoprotein. This kit includes the above fluorescent reagent; it also includes a buffer solution (such as 0.01 M PBS), and a human neutrophil apolipoprotein antibody protein. The concentrations of both fluorescent reagent I and fluorescent reagent II are 0.08 - 0.12 μg / mL, preferably 0.1 μg / mL.

[0007] In addition, the present invention also provides a method for preparing the above-mentioned human neutrophil apolipoprotein antibody fluorescent microsphere complex, comprising the following steps: Prepare fluorescent microspheres: Select the above-mentioned fluorescent microspheres with a particle size of 380 - 450 nm, having an excitation wavelength of 345 nm and emission wavelengths of 445 nm and 615 nm.

[0008] Antibody preparation: Use the human neutrophil apolipoprotein antibody purified by affinity chromatography, adjust the antibody concentration to about 1 mg / ml, and verify its specific binding activity by the indirect ELISA method before use.

[0009] Activation of the microsphere surface: Centrifuge the fluorescent microsphere suspension, discard the supernatant, resuspend with PBS and centrifuge again, then add EDC solution and NHS solution, and gently stir and react at room temperature for 30 minutes to convert the carboxyl groups on the microsphere surface into active esters.

[0010] Coupling reaction: Centrifuge the activated fluorescent microspheres, discard the supernatant, resuspend with PBS and then add the human neutrophil apolipoprotein antibody solution, and slowly stir overnight at 4°C to cause covalent binding between the amino groups of the antibody and the active esters on the microsphere surface.

[0011] Blocking: Centrifuge the fluorescent microspheres after the coupling reaction, discard the supernatant, resuspend with PBS and then add BSA solution, and gently stir and react at room temperature for 1 hour to block the unreacted active sites.

[0012] Washing and preservation: Centrifuge the blocked fluorescent microspheres, resuspend with PBS and repeat the centrifugation and resuspension steps to remove impurities. Finally, store the fluorescent microspheres conjugated with human neutrophil apolipoprotein antibody in PBS buffer at 4°C and add sodium azide as a preservative.

[0013] Among them, the concentrations of the EDC solution and the NHS solution are 10 mg / ml and 5 mg / ml respectively; the concentration of the BSA solution is 10% (w / v); the concentration of the preservative sodium azide is 0.02% (w / v).

[0014] Furthermore, the present invention also provides a method for detecting human neutrophil apolipoprotein using the human neutrophil apolipoprotein antibody fluorescent microsphere complex, comprising the following steps: Take fresh human venous whole blood, and take the supernatant serum after centrifugation; Mix the serum with PBS and the human neutrophil apolipoprotein antibody fluorescent microsphere complex, and oscillate and mix evenly; Put the mixture into a detection device for detection to obtain a fluorescence value, and calculate the protein concentration of the analyte according to the fluorescence value.

[0015] The beneficial effects of the present invention are: The present invention uses two different types of fluorescent substances. In the liquid phase, the two fluorescent substances are encapsulated together in microspheres based on polymethyl methacrylate. The two fluorescent substances do not interfere with each other. Their excitation lights are all around 345 nm, while the emission lights are around 445 nm and 615 nm, and the light at 615 nm occurs later than the light at 445 nm, with the interval at the microsecond level. This design gives play to the advantages of the light at 445 nm, which has high energy, high resolution, and high sensitivity; and the light at 615 nm, which has time-resolved ability and strong anti-interference ability. Two groups of CCD sensors respectively distinguish and record the signal intensities of the lights at 445 nm and 615 nm, and further improve the sensitivity and accuracy of the measurement system through calculation.

[0016] Common fluorescence detection methods use a single fluorescence signal. The excitation light and emission light wavelengths of Fluorescent Reagent II are close to each other, and background signal interference is likely to occur in practical applications. However, its emission light has a short wavelength and strong energy, and the advantages of the CCD capturing strong signals and having strong resolution. For rare-earth fluorescent substances, such as Fluorescent Reagent I which is a complex of europium, the separation degree between its excitation light and emission light wavelengths is high, and it has a microsecond-level delay effect, which can well avoid background interference. However, its emission light has a long wavelength and low energy, and the CCD signal is weak, resulting in low system sensitivity.

[0017] Using a single-excitation dual-emission system can make the advantages of the two fluorescent substances complementary to each other and improve the system resolution and precision. The two fluorescent substances in this system have the same excitation conditions, and their emission lights do not interfere with each other. One has strong resolution, and the other has a time-delay effect and strong anti-background interference ability. Moreover, the two fluorescent substances can be simultaneously coated in microspheres by polymethyl methacrylate, and then form an antibody-fluorescent microsphere complex after binding with antibody proteins. When the complex undergoes antigen-antibody binding with the protein to be detected, after the complex is centrifuged and separated, and irradiated by the 345-nm excitation light, its emission light is captured by two independent CCD detectors, and after passing through the calculation system, the light intensity values that can be mutually corrected are obtained, and the total amount of the substance to be detected can be obtained by referring to the standard curve. The single-excitation dual-emission system of the present invention has extremely important clinical application value. Description of the Drawings

[0018] Figure 1 It is the spectrum display of Example 2; Figure 2 It is the schematic diagram of the detection device. Detailed Embodiments

[0019] The present invention will be further described below in conjunction with the drawings. The following embodiments are only used to more clearly illustrate the technical solutions of the present invention and cannot be used to limit the protection scope of the present invention.

[0020] Example 1: A fluorescent reagent for the detection of human neutrophil apolipoprotein, comprising fluorescent reagent I and fluorescent reagent II, with the structural formulas as follows:

[0021] Conjugation of fluorescent microspheres and antibodies 1. Materials Fluorescent microspheres: Fluorescent microspheres with a particle size of 380 - 450 nm are selected, with an excitation wavelength of 345 nm and emission wavelengths of 445 and 615 nm.

[0022] Antibody preparation: Human neutrophil apolipoprotein antibody purified by affinity chromatography is used. The antibody concentration is adjusted to about 1 mg / ml, and its specific binding activity is verified by the indirect ELISA method before use.

[0023] Reagents: Phosphate - buffered saline (PBS, pH 7.4): As the basic reaction buffer, its formula is 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4.

[0024] 1 - Ethyl - 3 - (3 - dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N - hydroxysuccinimide (NHS) are respectively prepared into aqueous solutions of 10 mg / ml and 5 mg / ml, and are used immediately after preparation.

[0025] 10% (w / v) bovine serum albumin (BSA) solution is used to block unreacted sites.

[0026] 2. Activation of the microsphere surface Take 1 ml of the fluorescent microsphere suspension (microsphere concentration is about 1% w / v), centrifuge at 10000 rpm for 10 minutes, and discard the supernatant.

[0027] Resuspend the microspheres with 1 ml of PBS buffer, and centrifuge again to discard the supernatant.

[0028] Add 900 μl of PBS buffer to the precipitated microspheres, then sequentially add 100 μl of EDC solution and 50 μl of NHS solution, and gently stir (about 100 rpm) at room temperature for 30 minutes. During the reaction, EDC and NHS convert the carboxyl groups on the microsphere surface into active esters, making it easier to react with the amino groups of the antibody.

[0029] 3. Conjugation reaction After the reaction is completed, centrifuge the above - activated microspheres at 10000 rpm for 10 minutes, and discard the supernatant.

[0030] Resuspend the microspheres with 900 μl of PBS buffer, then add 100 μl of human neutrophil apolipoprotein antibody solution (1 mg / ml), and stir slowly (about 50 rpm) overnight at 4°C. During this process, covalent bonding occurs between the amino groups of the antibody and the activated groups on the surface of the microspheres to form a conjugate.

[0031] 4. Blocking After the conjugation reaction is completed, centrifuge the reaction system at 10,000 rpm for 10 minutes and discard the supernatant.

[0032] Resuspend the microspheres with 1 ml of PBS buffer, then add 100 μl of 10% BSA solution, and gently stir (about 100 rpm) at room temperature for 1 hour. This step is to block the unreacted active sites on the surface of the microspheres and reduce non-specific binding.

[0033] 5. Washing and storage After blocking, centrifuge the microspheres at 12,000 rpm for 15 minutes and discard the supernatant.

[0034] Resuspend the microspheres with 1 ml of PBS buffer, and repeat the centrifugation and resuspension steps 3 times to thoroughly remove the uncoupled antibodies and other impurities.

[0035] Finally, store the fluorescent microspheres conjugated with human neutrophil apolipoprotein antibody in PBS buffer at 4°C and add 0.02% (w / v) sodium azide as a preservative.

[0036] Thus, the fluorescent microsphere complex of human neutrophil apolipoprotein antibody is obtained.

[0037] Example 2: Fresh human venous whole blood is centrifuged at 3000 rpm for 15 minutes. Take 20 μl of the supernatant serum and add it to a colorimetric tube. Add 3 ml of PBS and 15 μl of the fluorescent microsphere complex of human neutrophil apolipoprotein antibody, oscillate and mix well for 30 s, and place it in the occlusion slot of the detection device.

[0038] Start the detection device. The schematic diagram of the detection device is as Figure 2As shown, the device emits excitation light from a computer-controlled LED. After passing through a filter, pure light of around 340 nm is obtained. Through a semi-reflective and semi-transmissive lens, part of the light reaches CCD detector 1, which detects the 340-nm light. This detection is an internal control of the light source to ensure that the light source is pure and the light intensity meets the system requirements. Another part of the 340-nm excitation light is focused on the sample detection chamber through a lens system to irradiate the sample. After excitation, the sample emits light at 445 nm and 615 nm successively. The corresponding CCD detectors of the system detect the light intensity values at 445 nm and 615 nm respectively, and convert them into electrical signals, which are transmitted to the computer software by the signal acquisition and analysis system for output. Insert a USB flash drive to read the reference curve, compare the curve with the measured light intensity, and calculate the protein concentration value.

[0039] Start the detection to obtain the reading and print the reading.

[0040] As Figure 1 shown, the filter group and the lens group system are jointly developed by Beijing Xintian Technology Co., Ltd. Filter performance: central wavelength: 340 ± 2 nm, transmittance: T ≥ 86%, cut-off wavelength: 260 - 384 nm, cut-off depth: OD6. The reading includes: the projection ratio (vertical axis) value of the first peak's abscissa from 260 - 384 nm from left to right is greater than 20, within the effective range. The extreme value of the projection ratio of the first peak is nearly 90, corresponding to the wavelength of 340 nm. The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the technical principle of the present invention, several improvements and deformations can still be made, and these improvements and deformations should also be regarded as the protection scope of the present invention.

Claims

1. A fluorescent reagent for detecting human neutrophil apolipoprotein, characterized in that: The fluorescent reagent includes fluorescent reagent I and fluorescent reagent II, and the structural formula is as follows: 。 2. The fluorescent reagent for detecting human neutrophil apolipoprotein according to claim 1, characterized in that: Application in human neutrophil apolipoprotein detection kit.

3. A human neutrophil apolipoprotein detection kit, characterized in that: The kit comprises the fluorescent reagent according to claim 1, wherein the concentration of the fluorescent reagent I is 0.08-0.12 μg / mL, and the concentration of the fluorescent reagent II is 0.08-0.12 μg / mL. The kit also comprises a buffer solution, which is 0.01 M PBS.

4. The human neutrophil apolipoprotein detection kit according to claim 3, characterized in that: The clinical specimens that can be detected by the human neutrophil apolipoprotein detection kit are fresh human venous blood, and human neutrophil apolipoprotein in anticoagulated or non-anticoagulated serum after centrifugation.

5. The human neutrophil apolipoprotein detection kit according to claim 3 or 4, characterized in that: The human neutrophil apolipoprotein detection kit also includes human neutrophil apolipoprotein antibody protein.

6. The human neutrophil apolipoprotein detection kit according to claim 3, characterized in that: The two fluorescent substances in the fluorescent reagent are encapsulated together in microspheres with polymethyl methacrylate as the base material in the liquid phase to form fluorescent microspheres. The two fluorescent substances will not interfere with each other, and their excitation lights are both around 345nm, and the emission lights are around 445nm and 615nm respectively, among which the emission light of 615nm is later than the emission light of 445nm, and the interval is at the microsecond level.

7. The human neutrophil apolipoprotein detection kit according to claim 6, characterized in that: The human neutrophil apolipoprotein detection kit also includes two independent CCD detectors for capturing emission light signals of 445nm and 615nm respectively, and obtaining light intensity values ​​that can be calibrated with each other through a calculation system, and obtaining the total amount of the neutrophil apolipoprotein to be detected by referring to the standard curve.

8. A method for preparing a human neutrophil apolipoprotein antibody fluorescent microsphere complex, characterized in that: The following steps are involved: a) preparing the fluorescent microspheres according to claim 6: selecting fluorescent microspheres with a particle size of 380-450 nm, an excitation wavelength of 345 nm, and emission wavelengths of 445 nm and 615 nm; b) Antibody preparation: Use human neutrophil apolipoprotein antibody purified by affinity chromatography, adjust the antibody concentration to about 1 mg / ml, and verify its specific binding activity by indirect ELISA method before use; c) Activation of microsphere surface: centrifuge the fluorescent microsphere suspension, discard the supernatant, resuspend with PBS and centrifuge again, then add EDC solution and NHS solution, gently stir and react at room temperature for 30 minutes to convert the carboxyl groups on the microsphere surface into active esters; d) Coupling reaction: centrifuge the activated fluorescent microspheres, discard the supernatant, resuspend them in PBS, add the human neutrophil apolipoprotein antibody solution, and slowly stir overnight at 4°C to allow the amino groups of the antibody to covalently bind to the active esters on the surface of the microspheres; e) Blocking: Centrifuge the fluorescent microspheres after the coupling reaction, discard the supernatant, resuspend them in PBS, add BSA solution, and gently stir the reaction at room temperature for 1 hour to block the unreacted active sites; f) Washing and storage: The blocked fluorescent microspheres were centrifuged, resuspended in PBS and repeated the centrifugation and resuspension steps to remove impurities. Finally, the fluorescent microspheres coupled to human neutrophil apolipoprotein antibodies were stored in PBS buffer at 4°C and sodium azide was added as a preservative.

9. The preparation method according to claim 8, characterized in that: The concentrations of the EDC solution and the NHS solution were 10 mg / ml and 5 mg / ml, respectively; the concentration of the BSA solution was 10% (w / v); and the concentration of the preservative sodium azide was 0.02% (w / v).

10. A method for detecting human neutrophil apolipoprotein using a human neutrophil apolipoprotein antibody-fluorescent microsphere complex, characterized in that: The human neutrophil apolipoprotein antibody fluorescent microsphere complex is prepared by the method of claim 8 or 9, comprising the following steps: 1) Take fresh human venous whole blood and obtain the supernatant serum after centrifugation; 2) Mix the serum with PBS and human neutrophil apolipoprotein antibody fluorescent microsphere complex and shake to mix; 3) The mixture is placed in a detection device for detection to obtain a fluorescence value, and the protein concentration of the detected object is calculated based on the fluorescence value.

Images