Application of a styrene-maleic anhydride copolymer and its composite

Abstract

The present invention relates to the application of styrene-maleic anhydride copolymers and their composites; during the application process, the stability of enzymes and the solid-phase carriers have an impact on antibodies, enzyme activity, and detection. For example, the surface characteristics of magnetic bead particles have an impact on both enzyme activity and detection. Styrene-maleic anhydride copolymers can be used as surface functional groups on chips to bind biological macromolecules and can be used as an ideal photosensitive material in surface plasmon resonance. The present invention can improve the activity and stability of labeled bioenzymes through solid-phase carriers, thereby improving detection sensitivity and reducing detection costs.

Description

Technical Field

[0001] The present invention relates to the field of chemiluminescence enzyme immunoassay, and particularly to the application of a styrene-maleic anhydride copolymer and its complex. Background Art

[0002] Chemiluminescence Enzyme Immunoassay (CLEIA) uses an enzyme involved in catalyzing a chemiluminescence reaction, such as horseradish peroxidase or alkaline phosphatase, to label an antigen or antibody. After an immune reaction with the corresponding antigen (antibody) in the test specimen, a magnetic bead-coated antibody - test antigen - enzyme-labeled antibody complex is formed. After washing, a luminescent agent is added, and the enzyme catalyzes and decomposes the substrate to emit light, and the concentration of the analyte is calculated. CLEIA combines the highly sensitive chemiluminescence measurement technology with the highly specific immune reaction and is used for the detection and analysis of various antigens, antibodies, hormones, enzymes, fatty acids, vitamins, drugs, etc. It is a new immunoassay technology developed after radioimmunoassay, enzyme immunoassay, fluorescence immunoassay, and time-resolved fluorescence immunoassay. CLEIA provides an ultra-trace immune detection means for scientific research, drug development, disease diagnosis, efficacy evaluation, food analysis and detection, etc.

[0003] Due to the influence of the biological activity of the labeled enzyme, the stability of the enzyme, and the solid-phase carrier on the enzyme activity and detection during the application of CLEIA. Previous studies have focused on related chemical reagents and reaction solvents, while there are few studies on the influence of the surface characteristics of solid-phase carriers, such as magnetic bead particles, on enzyme activity and detection. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of a styrene-maleic anhydride copolymer and its complex that can improve the detection sensitivity and reduce the detection cost.

[0005] To achieve the above purpose, the present invention provides a method for applying a styrene-maleic anhydride copolymer and its complex, which is an application in chemiluminescence enzyme immunoassay.

[0006] For the application of a styrene-maleic anhydride copolymer and its complex provided by the present invention, the degree of polymerization of the styrene-maleic anhydride copolymer and its complex is 25,000 - 118,000 MW.

[0007] For the application of a styrene-maleic anhydride copolymer and its complex provided by the present invention, the degree of polymerization of the styrene-maleic anhydride copolymer and its complex is about 89,000 MW.

[0008] Regarding the application of a styrene-maleic anhydride copolymer and its composite provided by the present invention, the concentration of the solution prepared from the styrene-maleic anhydride copolymer and its composite is 3-4%.

[0009] Regarding the application of a styrene-maleic anhydride copolymer and its composite provided by the present invention, the pH value of the solution prepared from the styrene-maleic anhydride copolymer and its composite is between 4 and 5.

[0010] Regarding the application of a styrene-maleic anhydride copolymer and its composite provided by the present invention, the inhibitor in the chemiluminescent enzyme immunoassay is diethylamine or its structural analog with a concentration of 1% and a dosage of 10-20 μl.

[0011] Regarding the application of a styrene-maleic anhydride copolymer and its composite provided by the present invention, the non-ionic surfactant in the chemiluminescent enzyme immunoassay is 10% Triton X-100 with a volume of 10-20 μl.

[0012] Regarding the application of a styrene-maleic anhydride copolymer and its composite provided by the present invention, the reaction temperature of the styrene-maleic anhydride copolymer and its composite in the chemiluminescent enzyme immunoassay is 30-35 °C, and the reaction time of the styrene-maleic anhydride copolymer and its composite in the chemiluminescent enzyme immunoassay is 30-40 min.

[0013] Regarding the application of a styrene-maleic anhydride copolymer and its composite provided by the present invention, the chemiluminescent enzyme immunoassay is a chemiluminescent enzyme immunoassay based on nanomagnetic beads as a solid-phase carrier.

[0014] Regarding the application of a styrene-maleic anhydride copolymer and its composite provided by the present invention, the concentration of the nanomagnetic beads is 20-30 μl of nanomagnetic beads with a concentration of 10 mg / ml.

[0015] In summary, the present invention has the following advantages:

[0016] In the application process of the styrene-maleic anhydride copolymer and its composite in chemiluminescent enzyme immunoassay, the stability of the enzyme and the influence of the solid-phase carrier on enzyme activity and detection are affected. For example, the surface characteristics of the solid-phase carrier magnetic bead particles have an impact on enzyme activity and detection. The styrene-maleic anhydride copolymer can be used as a surface functional group on the chip to bind biological macromolecules and can be used as an ideal photosensitive material in surface plasmon resonance. The present invention can improve the surface attachment degree of antibodies, the affinity of labeled enzymes, the stability of the three-dimensional space of the detection complex, and the reaction microenvironment through the solid-phase carrier, thereby enhancing the activity and stability of the labeled biological enzyme, and further improving the detection sensitivity and reducing the detection cost.

[0017] The above description is only an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and be implemented in accordance with the content of the specification, the following provides a detailed description of the preferred embodiments of the present invention as follows. Brief Description of the Drawings

[0018] Figure 1 It is a schematic structural diagram of a styrene-maleic anhydride copolymer in the application of a styrene-maleic anhydride copolymer and its composite provided by the present invention;

[0019] Figure 2 It is a schematic diagram of the degree of polymerization of SMA and the detection signal in an embodiment of the application of a styrene-maleic anhydride copolymer and its composite provided by the present invention;

[0020] Figure 3 It is a schematic diagram of the concentration of SMA and the detection signal in an embodiment of the application of a styrene-maleic anhydride copolymer and its composite provided by the present invention;

[0021] Figure 4 It is a schematic diagram of the pH value of the SMA solution and the detection signal in an embodiment of the application of a styrene-maleic anhydride copolymer and its composite provided by the present invention;

[0022] Figure 5 It is a schematic diagram of the dosage of nano magnetic beads and the detection signal in an embodiment of the application of a styrene-maleic anhydride copolymer and its composite provided by the present invention;

[0023] Figure 6 It is a schematic diagram of the dosage of the polymerization inhibitor diethylamine and the detection signal in an embodiment of the application of a styrene-maleic anhydride copolymer and its composite provided by the present invention;

[0024] Figure 7 It is a schematic diagram of the dosage of the non-ionic surfactant Triton 100 and the detection signal in an embodiment of the application of a styrene-maleic anhydride copolymer and its composite provided by the present invention;

[0025] Figure 8 It is a schematic diagram of the reaction temperature and the detection signal in an embodiment of the application of a styrene-maleic anhydride copolymer and its composite provided by the present invention;

[0026] Figure 9 It is a schematic diagram of the reaction time and the detection signal in an embodiment of the application of a styrene-maleic anhydride copolymer and its composite provided by the present invention. Detailed Embodiments

[0027] The following combines the embodiments and the drawings to further describe in detail the specific embodiments of the present invention. The following embodiments are used to illustrate the present invention, but are not used to limit the scope of the present invention.

[0028] Example

[0029] 1. Modify the nano magnetic beads with SMA, and then conjugate the capture antibody;

[0030] 2. React the magnetic bead antibody from step 1 + sample + enzyme-labeled antibody at 35 °C for 15 min;

[0031] 3. Wash twice;

[0032] 4. React the substrate at 35 °C for 15 min;

[0033] 5. Read the value on the chemiluminescence detector.

[0034] In this example, the degree of polymerization of SMA has a great influence on chemiluminescence. The degrees of polymerization of different SMAs are selected as 6000WM, 9100WM, 25000WM, 89000MW, 118000MW, and 100000 - 500000MW respectively. Different degrees of polymerization have different effects on the surface attachment degree of antibodies, the affinity of labeled enzymes, the stability of the three-dimensional space of the detection complex, and the reaction microenvironment. Due to their low degrees of polymerization, 6000WM and 9100WM cannot firmly attach antibodies to the solid-phase carrier, so an effective enzymatic three-dimensional space cannot be formed, and an effective enzymatic reaction cannot be constituted. The SMA polymer with a degree of polymerization of 100000 - 500000MW has too high a degree of polymerization, which easily forms steric hindrance, making the stability of attachment and binding and the microenvironment of the labeled enzyme unfavorable for the enzymatic reaction. The relatively ideal degree of polymerization is from 25000MW to 118000MW, and the optimal degree of polymerization is about 89000MW, see Figure 2 .

[0035] In this example, the concentration of SMA with a degree of polymerization of 89000MW affects the thickness of SMA on the surface of the solid-phase carrier, the formation of the three-dimensional space structure, the density of functional groups, and the photosensitivity intensity to a certain extent. Optical signals can be detected on the surface of the solid-phase carrier treated with 1% - 8% SMA. Too low or too high a concentration is not conducive to the detection of CLEIA, and the optimal SMA concentration is 3 - 4%, see Figure 3 .

[0036] In this example, optical signals can be detected when the pH value of the SMA solution ranges from 3.5 to 9. However, when the pH is higher than 5, the detection signal decreases with the increase of the pH value. When the pH value is higher than 7, it will significantly affect the stability of the anhydride group, thereby affecting the binding ability of the antibody and the detection signal. The optimal pH value is between 4 and 5, see Figure 4 .

[0037] In this embodiment, it is necessary to determine the optimal dosage of nanomagnetic beads. From the experimental results, it can be seen that 10 to 50 ul of 10 mg / ml nanomagnetic beads can give a signal. Considering the cost and detection sensitivity, it is recommended that the dosage of nanomagnetic beads is 20 to 30 ul of 10 mg / ml. Figure 5 .

[0038] In this embodiment, the inhibitor diethylamine or its structural analogue can effectively bind to the excess anhydride groups in the SMA polymer, thereby reducing nonspecific binding and increasing the specificity and sensitivity of the detection. Moreover, due to the addition of diethylamine, the excess anhydride groups in the SMA can be effectively opened, thereby increasing the hydrophobicity of the carrier surface. From the experimental results, it can be seen that 10ul and above 1% diethylamine can effectively enhance the chemiluminescent signal. Figure 6 There is no obvious difference in the chemical signals detected after treatment with 10-50ul diethylamine, indicating that the excess anhydride groups in SMA are fully opened. Considering the detection cost and effectiveness, it is recommended to use 10-20ul of 1% diethylamine.

[0039] In this embodiment, the surface detergent SDS, the emulsifier Tween 20 and the non-ionic surfactant Triton 100 have different effects on the detection signal. When the volume used is higher than 20ul, the detection signal intensity is reduced. Low doses are conducive to detecting the signal. Among them, the signal enhancement effect of the non-ionic surfactant Triton 100 is the best, see Figure 7 .

[0040] In this embodiment, reaction temperature is one of the important factors that determine the bioenzymatic reaction. The test results show that the signal can be detected at a reaction temperature of 25 to 45 degrees. The optimal temperature is 30 to 35 degrees. Figure 8 .

[0041] In this embodiment, a significant signal can be detected starting from 10 minutes of reaction time, and the peak value is close to 30 minutes. Considering the effectiveness of the detection, the recommended reaction time is about 30 to 40 minutes. Figure 9 .

[0042] In the application process of styrene-maleic anhydride copolymer and its composites in chemiluminescent enzyme immunoassay, the stability of the enzyme and the solid-phase carrier have an impact on both enzyme activity and detection. For example, the surface characteristics of the solid-phase carrier magnetic bead particles have an impact on enzyme activity and detection. Styrene-maleic anhydride copolymer can be used as a surface functional group on the chip to bind biological macromolecules and can be used as an ideal photosensitive material in surface plasmon resonance. In the present invention, the solid-phase carrier modified with SMA (such as nanomagnetic beads) can improve the surface attachment degree of antibodies, the affinity of labeled enzymes, the three-dimensional space stability of the detection complex, and the reaction microenvironment, thereby enhancing the activity and stability of labeled bioenzymes, and further improving the detection sensitivity and reducing the detection cost.

[0043] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. It should be pointed out that for those of ordinary skill in the art in this technical field, without departing from the technical principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

[0044] In all descriptions of the present invention, it should be understood that unless otherwise defined, the terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. It should be further understood that the terms should be interpreted as having a meaning consistent with their meaning in the relevant art and the context of the present invention, and should not be interpreted in an idealized or overly formalized sense unless clearly defined herein.

Claims

1. Application of a styrene-maleic anhydride copolymer, characterized in that: Application of styrene-maleic anhydride copolymer in chemiluminescent enzyme immunoassay, wherein the degree of polymerization of the styrene-maleic anhydride copolymer is 25,000 - 118,000 MW.

2. The application of the styrene-maleic anhydride copolymer according to claim 1, wherein: The degree of polymerization of the styrene-maleic anhydride copolymer is about 89,000 MW.

3. Use of the styrene-maleic anhydride copolymer according to claim 1, characterized in that: The concentration of the solution prepared from the styrene-maleic anhydride copolymer is 3 - 4%.

4. Use of the styrene-maleic anhydride copolymer according to claim 1, characterized in that: The pH value of the solution prepared from the styrene-maleic anhydride copolymer is between 4 and 5.

5. Use of the styrene-maleic anhydride copolymer according to claim 1, characterized in that: The inhibitor in the chemiluminescent enzyme immunoassay is diethylamine with a concentration of 1% and a dosage of 10 - 20 μl.

6. Use of the styrene-maleic anhydride copolymer according to claim 1, characterized in that: The non-ionic surfactant in the chemiluminescent enzyme immunoassay is 10% Triton X-100 with a volume of 10 - 20 μl.

7. Use of the styrene-maleic anhydride copolymer according to claim 1, characterized in that: The reaction temperature of the styrene-maleic anhydride copolymer in the chemiluminescent enzyme immunoassay is 30 - 35 °C, and the reaction time of the styrene-maleic anhydride copolymer in the chemiluminescent enzyme immunoassay is 30 - 40 min.

8. Use of the styrene-maleic anhydride copolymer according to claim 1, characterized in that: The chemiluminescent enzyme immunoassay is a chemiluminescent enzyme immunoassay based on nanomagnetic beads as a solid-phase carrier.

9. Use of the styrene-maleic anhydride copolymer according to claim 8, characterized in that: The nanomagnetic beads are 20 - 30 μl of nanomagnetic beads with a concentration of 10 mg / ml.

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