Surface plasma resonance chip for detecting tetanus antibody concentration in serum as well as preparation method and application of surface plasma resonance chip

Abstract

The invention provides a surface plasma resonance chip for detecting tetanus antibody concentration in serum and a preparation method and application thereof, and belongs to the technical field of medicine detection. The chip for detecting the tetanus antibody concentration in the serum, provided by the invention, is a surface plasma resonance chip coupled with tetanus toxoid, and the coupling level of the tetanus toxoid is 1,000 RU to 10,000 RU. According to the chip and the detection method provided by the invention, the concentration level of the tetanus antibody in a serum sample can be specifically and quantitatively detected, the detection sensitivity is high, the linear range is wide, the precision and the accuracy are good, and the requirements of quantitative analysis of biological samples can be met.

Description

Technical Field

[0001] The invention belongs to the technical field of medical detection, and in particular relates to a surface plasmon resonance chip for detecting tetanus antibody concentration in serum, and a preparation method and application thereof. Background Art

[0002] Tetanus is an acute infectious disease caused by the spores of Clostridium tetani. The best way to prevent tetanus is to vaccinate on time throughout the life cycle and to achieve and maintain an appropriate level of booster immunization. The main indicator for assessing the need for booster immunization is the concentration of protective antibodies in the body.

[0003] The acceptable level of circulating anti-tetanus antibodies required to measure protection in in vivo toxin neutralization tests is 0.01 IU / ml. When using in vitro methods such as standard enzyme-linked immunosorbent assay (ELISA), antibody level readings of at least 0.1–0.2 IU / ml are considered protective. The standard ELISA method is the most commonly used method to assess tetanus antibody levels, however, this method has disadvantages associated with long run times, indirect formats, laboriousness, high costs, and the need for multiple washing steps. The cumbersome operating steps also lead to low accuracy and precision of the ELISA method.

[0004] Surface plasmon is an electromagnetic wave on the surface of a metal, generated by the interaction of freely vibrating photons and electrons. Surface plasmon resonance (SPR) is an optical phenomenon that occurs on the surface of two media and can be induced by photons or electrons. When light is emitted from a dense medium into a sparse medium, it undergoes total reflection, forming an evanescent wave that enters the sparse medium. When the totally reflected evanescent wave meets the plasma wave on the metal surface, resonance may occur, the energy of the reflected light decreases, and a resonance peak appears in the energy spectrum of the reflected light. This resonance is called surface plasmon resonance. The incident angle that causes surface plasmon resonance is called the SPR angle. The SPR biosensor is an optical sensor that can detect the binding of biomolecules on the metal surface by detecting changes in the near-surface refractive index of the gold film. SPR technology is a powerful tool for studying the interaction of biomolecules, but there are currently few reports on the application of the SPR method in clinical testing. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a surface plasmon resonance chip for detecting the concentration of tetanus antibodies in serum, which can specifically and quantitatively detect the concentration level of tetanus antibodies in serum samples.

[0006] The present invention also provides a method for detecting the concentration of tetanus antibodies in serum, which can quantitatively detect the concentration of tetanus antibodies in human serum, has high detection sensitivity, wide linear range, good precision and accuracy, and can meet the requirements of quantitative analysis of biological samples.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a chip for detecting the concentration of tetanus antibodies in serum. The chip is a surface plasmon resonance chip coupled with tetanus toxoid, and the coupling level of the tetanus toxoid is 1000 response units (RU) to 10000 response units (RU).

[0008] The present invention also provides a method for preparing the above-mentioned chip, comprising the following steps: first injecting EDC / NHS onto a surface plasmon resonance chip fixed with carboxymethyl dextran; then injecting a tetanus toxoid solution to make the coupling level of the tetanus toxoid be 1000RU~10000RU; sealing to obtain the chip; the concentration of tetanus toxoid in the tetanus toxoid solution is 24μg / ml~60μg / ml; the pH value of the tetanus toxoid solution is 4.0~5.0.

[0009] Preferably, the flow rate of the tetanus toxoid solution is 5 μl / min and the injection time is 20 min.

[0010] The present invention also provides the use of the chip or the chip prepared by the preparation method in preparing a product for detecting the concentration of tetanus antibodies in serum.

[0011] The present invention also provides a kit for detecting the concentration of tetanus antibodies in serum, wherein the kit comprises the above chip or the chip prepared by the above preparation method.

[0012] Preferably, the kit further comprises a blank matrix, a tetanus human immunoglobulin standard and a regeneration solution; the blank matrix is ​​tetanus antibody negative serum.

[0013] The present invention also provides a method for detecting the concentration of tetanus antibodies in serum, comprising the following steps: First, a blank matrix is ​​passed through the surface of the chip, and then tetanus human immunoglobulin standard solutions of different concentrations are passed through the surface of the chip to obtain response signals 1 of different concentrations; the response signal 1 = the response signal of the tetanus human immunoglobulin standard solution - the response signal of the blank matrix; A standard curve was prepared with the concentration of tetanus human immunoglobulin standard solution as the abscissa and the response signal 1 of different concentrations as the ordinate; After the chip is cleaned with a regeneration solution, a blank matrix is ​​first passed through the surface of the chip, and then a serum sample to be tested is passed through the surface of the chip to obtain a response signal 2; the response signal 2 = the response signal of the serum sample to be tested - the response signal of the blank matrix; The response signal 2 is substituted into the standard curve to calculate the tetanus antibody concentration in the serum sample to be tested.

[0014] Preferably, the standard curve is y=ax+b, R 2 >0.99, the x is the tetanus antibody concentration, the y is the response signal, the a>500, and the absolute value of the b is <3.

[0015] Preferably, the regeneration solution is a glycine solution, and the pH of the glycine solution is 1.5.

[0016] Preferably, the step of cleaning with the regeneration solution is: injecting the regeneration solution into the chip at a flow rate of 30 μl / min, the regeneration time is 120 s, and the regeneration is stabilized for 80 s.

[0017] Beneficial effects of the present invention: The surface plasmon resonance chip provided by the present invention can specifically and quantitatively detect the concentration level of tetanus antibodies in serum samples. Compared with the traditional ELISA method, the detection method provided by the present invention has better sensitivity and a wider linear range, and the lower limit of quantification is 0.001 IU / ml. In addition, the method for detecting the concentration of tetanus antibodies in serum provided by the present invention is a program injection method, which has higher precision and accuracy than the ELISA method of well plate determination. The method provided by the present invention can overcome the adverse effects of complex substances in serum samples on the accuracy of the final test results, can eliminate the influence of nonspecific adsorption in serum samples, and thus can quickly, accurately and sensitively detect the level of tetanus antibody concentration in serum samples.

[0018] The present invention carries out methodological verification of the method for detecting tetanus antibody concentration in serum provided by the present invention according to the requirements of ICH M10. The results show that the detection method provided by the present invention has good precision and accuracy, and has higher automation and accuracy than the ELISA method, thus providing a feasible method for serological surveys of large populations with large samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The result diagram of Example 3, wherein the left diagram is the sensor diagram of the surface plasmon resonance chip obtained in Example 1, and the right diagram is the corresponding sensor diagram of 0.001 IU / ml tetanus human immunoglobulin national standard solution; Figure 2The result diagram of Example 4, wherein the left diagram is the sensor diagram of the surface plasmon resonance chip obtained in Example 2, and the right diagram is the corresponding sensor diagram of 0.001 IU / ml tetanus human immunoglobulin national standard solution; Figure 3 The result diagram of Comparative Example 2, wherein the left diagram is the sensor diagram of the surface plasmon resonance chip obtained in Comparative Example 1, and the right diagram is the sensor diagram of the corresponding 0.001 IU / ml tetanus human immunoglobulin national standard solution; Figure 4 A standard curve diagram for calibration of the tetanus human immunoglobulin standard of the present invention; Figure 5 This is the sensorgram regenerated in 10 mM hydrochloric acid solution; Figure 6 This is the sensorgram regenerated with 10 mM NaOH solution; Figure 7 This is the sensor diagram of glycine hydrochloride regeneration at pH 1.5; Figure 8 The sensorgram of the real serum sample measurement, where the red line represents the sensor curve of the test solution and the gray line represents the sensor curve of the blank matrix. DETAILED DESCRIPTION

[0020] The present invention provides a chip for detecting the concentration of tetanus antibodies in serum. The chip is a surface plasmon resonance chip coupled with tetanus toxoid, and the coupling level of the tetanus toxoid is 1000RU-10000RU.

[0021] In the present invention, the surface plasmon resonance chip coupled with tetanus toxoid is preferably a surface plasmon resonance chip fixed with carboxymethyl dextran. The present invention does not specifically limit the specific source of the surface plasmon resonance chip fixed with carboxymethyl dextran, and any conventional commercially available product in the art can be used. In the present invention, the carboxymethyl dextran on the surface plasmon resonance chip is activated by EDC / NHS and catalyzed to undergo an esterification reaction, and then covalently binds to the amino group of tetanus toxoid, thereby ensuring that the target protein is coupled to the gold foil surface. The present invention does not specifically limit the specific source of tetanus toxoid.

[0022] The present invention also provides a method for preparing the above-mentioned chip, comprising the following steps: first injecting EDC / NHS onto a surface plasmon resonance chip fixed with carboxymethyl dextran; then injecting a tetanus toxoid solution to make the coupling level of the tetanus toxoid be 1000RU~10000RU; sealing to obtain the chip; the concentration of tetanus toxoid in the tetanus toxoid solution is 24μg / ml~60μg / ml; the pH value of the tetanus toxoid solution is 4.0~5.0.

[0023] In the preparation method of the present invention, EDC / NHS is first injected into the surface plasmon resonance chip fixed with carboxymethyl dextran to activate the chip surface. In the present invention, the flow rate of injecting EDC / NHS is preferably 10μ1 / min, and the injection time is preferably 600s, so that the baseline is raised by 100~300RU. In the present invention, the injected tetanus toxoid solution is preferably a sodium acetate solution containing tetanus toxoid, and the pH value of the sodium acetate solution containing tetanus toxoid is preferably 4.2~4.8, and the concentration of tetanus toxoid in the tetanus toxoid solution is preferably 30μg / ml~50μg / ml, and more preferably 35μg / ml~45μg / ml. In the present invention, the flow rate of injecting tetanus toxoid solution is preferably 5μl / min, and the injection time is preferably 20min. In the present invention, the solution used for sealing is preferably ethanolamine hydrochloric acid, the flow rate of injecting ethanolamine hydrochloric acid is preferably 10 μl / min, and the injection time is preferably 420 s.

[0024] The present invention also provides the use of the above chip or the chip prepared by the above preparation method in preparing a product for detecting the concentration of tetanus antibodies in serum. In the present invention, the type of the product preferably includes a kit.

[0025] The present invention also provides a kit for detecting the concentration of tetanus antibodies in serum, wherein the kit comprises the above chip or the chip prepared by the above preparation method.

[0026] In the present invention, the kit preferably further comprises a blank matrix, a tetanus human immunoglobulin standard and a regeneration solution; the blank matrix is ​​tetanus antibody negative serum. The blank matrix is ​​preferably obtained by diluting tetanus antibody negative serum 10 times with an interaction buffer and filtering with a 0.2 μm filter membrane. The interaction buffer is preferably 1.0×PBS-P+ with a pH of 7.4. The regeneration solution is preferably a glycine solution, and the pH of the glycine solution is preferably 1.5.

[0027] The present invention also provides a method for detecting the concentration of tetanus antibodies in serum, comprising the following steps: First, a blank matrix is ​​passed through the surface of the chip, and then tetanus human immunoglobulin standard solutions of different concentrations are passed through the surface of the chip to obtain response signals 1 of different concentrations; the response signal 1 = the response signal of the tetanus human immunoglobulin standard solution - the response signal of the blank matrix; A standard curve was prepared with the concentration of tetanus human immunoglobulin standard solution as the abscissa and the response signal 1 of different concentrations as the ordinate; After the chip is cleaned with a regeneration solution, a blank matrix is ​​first passed through the surface of the chip, and then a serum sample to be tested is passed through the surface of the chip to obtain a response signal 2; the response signal 2 = the response signal of the serum sample to be tested - the response signal of the blank matrix; The response signal 2 is substituted into the standard curve to calculate the tetanus antibody concentration in the serum sample to be tested.

[0028] In the method of the present invention, the different concentrations of tetanus human immunoglobulin standard solutions are preferably prepared by diluting the tetanus human immunoglobulin standard with a blank matrix to different concentrations of 0.001 IU / ml, 0.005 IU / ml, 0.01 IU / ml, 0.02 IU / ml, 0.05 IU / ml and 0.1 IU / ml. In the present invention, the standard curve is preferably y=ax+b, R 2 >0.99, x is the tetanus antibody concentration, y is the response signal, a>500, the absolute value of b is<3; R 2 Preferably, it is 0.9992~0.9996, preferably, a is 1195~1300, and preferably, b is 0.6836~1.371.

[0029] In the present invention, the regeneration solution is preferably a glycine solution, and the pH of the glycine solution is preferably 1.5. The step of cleaning with the regeneration solution is preferably: injecting the regeneration solution into the chip at a flow rate of 30μl / min, the regeneration time is 120s, stabilizing for 80s after regeneration, and then testing the next sample. In the present invention, the binding time of the sample to be tested and the chip is preferably 100s, and then dissociating with the interaction buffer for 100s, and then cleaning with the regeneration solution. The flow rate of the interaction buffer dissociation is preferably 30μl / min.

[0030] The surface plasmon resonance method for detecting tetanus antibody concentration in serum provided by the present invention has the following advantages over traditional technologies such as fluorescence or ELISA: ① the analyzed substance can be directly used for detection without any labeling; ② the interaction between molecules can be monitored in real time; ③ the tetanus antibody concentration in human serum can be quantitatively detected with high detection sensitivity, wide linear range, good precision and accuracy, and can meet the requirements of quantitative analysis of biological samples.

[0031] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0032] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0033] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0034] The surface plasmon resonance instrument used in the following examples is Biacore T200, purchased from GE Healthcare; PBS-P+ was purchased from Cytiva; ultrapure water was purchased from Field (Beijing) Scientific Instrument Co., Ltd.; glycine hydrochloride solution (Glycine 1.5) with a pH of 1.5 was purchased from Cytiva; sodium acetate with a pH of 4.0 was purchased from Cytiva; amino coupling kit was purchased from Cytiva; tetanus toxoid was purchased from Creative Biolabs; tetanus human immunoglobulin national standard was purchased from China Food and Drug Inspection Institutes; tetanus antibody negative serum was purchased from Zhengzhou Yite Biotechnology Co., Ltd.; CMV antibody was purchased from Shanghai Tojing Life Science Co., Ltd.

[0035] The interaction buffer used in the following examples: 1.0×PBS-P+, pH 7.4, filtered through a 0.22 μm filter membrane and degassed; blank matrix: tetanus antibody negative serum was diluted 10 times with the interaction buffer and filtered through a 0.2 μm filter membrane.

[0036] Example 1 A surface plasmon resonance chip for detecting tetanus antibody concentration in serum, wherein the chip is a surface plasmon resonance chip coupled with 10000RU tetanus toxoid.

[0037] The preparation method is: Activate the chip surface: inject EDC / NHS (from the amino coupling kit, Cytiva) into the surface of a surface plasmon resonance chip (Cytiva) with carboxymethyl dextran immobilized on the gold foil surface, at a flow rate of 10 μl / min for 600 s, raising the baseline by 266 RU; Ligand coating: inject a 10 mM sodium acetate solution containing tetanus toxoid at a pH of 4.0, wherein the concentration of tetanus toxoid in the sodium acetate solution is 60 μg / ml, and the injection flow rate is 5 μl / min. Repeat the injection until the coupling level reaches 10,000 RU, then stop the injection. The injection time is 20 min.

[0038] Blocking: Inject 1M ethanolamine hydrochloric acid (from the amino coupling kit, Cytiva) at a flow rate of 10 μl / min for 420 s.

[0039] Example 2 A surface plasmon resonance chip for detecting tetanus antibody concentration in serum is disclosed. The chip is a surface plasmon resonance chip coupled with 1000RU tetanus toxoid.

[0040] The preparation method is: Activate the chip surface: inject EDC / NHS (from the amino coupling kit, Cytiva) into the surface of a surface plasmon resonance chip (Cytiva) with carboxymethyl dextran immobilized on the gold foil surface, at a flow rate of 10 μl / min for 600 s, raising the baseline by 562 RU; Ligand coating: inject a 10 mM sodium acetate solution containing tetanus toxoid at a pH of 5.0, wherein the concentration of tetanus toxoid in the sodium acetate solution is 24 μg / ml, and the injection flow rate is 5 μl / min. Repeat the injection until the coupling level reaches 1000RU, and then stop the injection.

[0041] Blocking: Inject 1M ethanolamine hydrochloric acid (from the amino coupling kit, Cytiva) at a flow rate of 10 μl / min for 420 s.

[0042] Comparative Example 1 A surface plasmon resonance chip for detecting tetanus antibody concentration in serum, wherein the chip is a surface plasmon resonance chip coupled with 500RU tetanus toxoid.

[0043] The preparation method is: Activate the chip surface: inject EDC / NHS (from the amino coupling kit, Cytiva) into the surface of a surface plasmon resonance chip (Cytiva) with carboxymethyl dextran immobilized on the gold foil surface, at a flow rate of 10 μl / min for 600 s, raising the baseline by 401 RU; Ligand coating: inject a 10 mM sodium acetate solution containing tetanus toxoid at a pH of 5.0, wherein the concentration of tetanus toxoid in the sodium acetate solution is 12 μg / ml, and the injection flow rate is 5 μl / min. Repeat the injection until the coupling level reaches 500RU, and then stop the injection.

[0044] Blocking: Inject 1M ethanolamine hydrochloric acid (from the amino coupling kit, Cytiva) at a flow rate of 10 μl / min for 420 s.

[0045] Example 3 First, a blank matrix was injected into a surface plasmon resonance instrument Biacore T200 (SPR detector), and the blank matrix was passed through the surface plasmon resonance chip obtained in Example 1. The blank matrix was obtained by diluting tetanus antibody negative serum 10 times with an interaction buffer and filtering with a 0.2 μm filter membrane. The interaction buffer was 1.0×PBS-P+, pH 7.4, filtered through a 0.22 μm filter membrane, and degassed.

[0046] Then, a 0.001 IU / ml tetanus human immunoglobulin national standard solution (the solution is obtained by diluting the blank matrix) is injected into a surface plasmon resonance instrument Biacore T200 (SPR detector), and the tetanus human immunoglobulin national standard solution is passed through the surface plasmon resonance chip obtained in Example 1.

[0047] The response signal reporting point was 80s after the injection, and the response signal of the tetanus human immunoglobulin national standard solution and the response signal of the blank matrix were obtained respectively. The response signal of the tetanus human immunoglobulin national standard solution - the response signal of the blank matrix = 1.4RU. Figure 1 .

[0048] Example 4 The difference from Example 3 is that the surface plasmon resonance chip in Example 3 is replaced by the surface plasmon resonance chip obtained in Example 2, and the rest is the same as Example 3. The results are shown in FIG. Figure 2 , the response signal of the tetanus human immunoglobulin national standard solution - the response signal of the blank matrix = 0.9RU.

[0049] Comparative Example 2 The difference from Example 3 is that the surface plasmon resonance chip in Example 3 is replaced by the surface plasmon resonance chip obtained in Comparative Example 1, and the rest is the same as Example 3. The results are shown in FIG. Figure 3 , the response signal of the tetanus human immunoglobulin national standard solution - the response signal of the blank matrix = 0.3RU.

[0050] From the results of Example 3, Example 4 and Comparative Example 2, it can be seen that the higher the chip coupling level, the greater the signal of the 0.001 IU / ml standard solution, that is, the better the sensitivity of the method. Therefore, when the method of the present invention is used for detection, tetanus toxoid should be saturatedly coupled.

[0051] Example 5 A kit for detecting tetanus antibody concentration in serum, the kit comprising the chip prepared in Example 1, a blank matrix and a tetanus human immunoglobulin standard; the blank matrix is ​​obtained by diluting tetanus antibody negative serum 10 times with an interaction buffer and filtering with a 0.2 μm filter membrane; the interaction buffer is 1.0×PBS-P+ with a pH of 7.4, filtered through a 0.22 μm filter membrane, and degassed.

[0052] Example 6 A method for detecting tetanus antibody concentration in serum comprises the following steps: firstly injecting a blank matrix (same as in Example 5) into a surface plasmon resonance instrument Biacore T200 (SPR detector), and allowing the blank matrix to flow through the surface of the chip obtained in Example 1; using the blank matrix to dilute the tetanus human immunoglobulin national standard into tetanus human immunoglobulin standard solutions of different concentrations of 0.001 IU / ml, 0.005 IU / ml, 0.01 IU / ml, 0.02 IU / ml, 0.05 IU / ml and 0.1 IU / ml, respectively, injecting the tetanus human immunoglobulin standard solutions of different concentrations into a surface plasmon resonance instrument Biacore T200 (SPR detector), and allowing the tetanus human immunoglobulin standard solutions of different concentrations to flow through the surface of the chip obtained in Example 1, and obtaining response signals 1 of different concentrations; the response signal 1=the response signal of the tetanus human immunoglobulin standard solution-the response signal of the blank matrix; A standard curve was prepared with the concentration of tetanus human immunoglobulin standard solution as the horizontal axis and the response signal 1 of different concentrations as the vertical axis. Figure 4 .

[0053] When the above-mentioned tetanus human immunoglobulin standard solutions of different concentrations flow through the chip obtained in Example 1, after each concentration flows through, it is necessary to use a regeneration solution of glycine hydrochloride (Glycine pH1.5) with a pH of 1.5 to clean the above-mentioned chip, and then flow through the next concentration of tetanus human immunoglobulin standard solution. The cleaning is: wait for the tetanus human immunoglobulin standard solution to bind to the detection chip for 100s, dissociate it with an interaction buffer (same as Example 5) for 100s, and the flow rate is 30μl / min; then use a regeneration solution of glycine hydrochloride (Glycine pH1.5) with a pH of 1.5 to inject the chip at a flow rate of 30μl / min for 120s, stabilize for 80s after the cleaning, and then flow through the next concentration of tetanus human immunoglobulin standard solution.

[0054] After the standard curve is prepared, the chip is dissociated with an interaction buffer (same as in Example 5) for 100 seconds at a flow rate of 30 μl / min; then, a regeneration solution of glycine hydrochloride (Glycine pH 1.5) with a flow rate of 30 μl / min is injected into the chip for 120 seconds. After washing, the chip is stabilized for 80 seconds. Then, a blank matrix is ​​passed through the chip surface, and then a serum sample to be tested is passed through the chip surface to obtain a response signal 2; the response signal 2 = the response signal of the serum sample to be tested - the response signal of the blank matrix; Substitute the response signal 2 into the above standard curve y=1300x+1.325, R 2=0.9994, wherein x is the tetanus antibody concentration, and y is the response signal 2, and the tetanus antibody concentration in the serum sample to be tested is obtained by calculation.

[0055] Example 7 Screening of regeneration solution The chip obtained in Example 1 through which the serum sample to be tested flowed was cleaned with three regeneration solutions, namely, 10mM NaOH solution, 10mM hydrochloric acid solution and glycine hydrochloric acid with a pH of 1.5. The cleaning process was as follows: the serum sample to be tested was combined with the chip obtained in Example 1 for 100s, and dissociated with the interaction buffer (same as Example 5) for 100s at a flow rate of 30μl / min; then different regeneration solutions were injected into the chip at a flow rate of 30μl / min for 120s, and stabilized for 80s after the cleaning. The advantages and disadvantages of different regeneration solutions were compared according to the degree to which the signal returned to the baseline. The results are shown in Figure 5-Figure 7 As shown, 10 mM hydrochloric acid solution regenerated vigorously, 10 mM NaOH solution regenerated incompletely, and glycine hydrochloric acid with a pH of 1.5 regenerated well.

[0056] Example 8 The detection method of Example 6 was methodologically validated with reference to Appendix 9012 of Part IV of the 2020 edition of the Chinese Pharmacopoeia for Validation of Quantitative Analysis Methods for Biological Samples and the ICH Coordinated Guidelines for Validation of Bioanalytical Methods and Sample Analysis M10.

[0057] (1) Specificity CMV antibody interferor solutions with concentrations of 0.001 IU / ml, 0.01 IU / ml and 0.1 IU / ml were prepared with blank matrix, and the tetanus human immunoglobulin national standard was diluted to 0.002 IU / ml (low concentration QC) and 0.08 IU / ml (high concentration QC) with the above-prepared CMV antibody interferor solutions with three concentrations of low, medium and high. The blank matrix, a series of calibration curve solutions (the series of calibration curve solutions are the series of calibration curve solutions in which the tetanus human immunoglobulin national standard is diluted to 0.001 IU / ml, 0.005 IU / ml, 0.01 IU / ml, 0.02 IU / ml, 0.05 IU / ml and 0.1 IU / ml with blank matrix), CMV antibody interferor solution and QC solution were injected into the SPR detector (the chip used was obtained in Example 1). The experimental results showed that the responses of the matrix after adding CMV antibody were consistent with those of the blank matrix; the recoveries of the low-concentration QC and high-concentration QC prepared with CMV antibody interferor solution were both in the range of 80%~120%, see Table 1.

[0058] Table 1 Specificity results

[0059] (2) Calibration curve and range The tetanus human immunoglobulin national standard was diluted with a blank matrix into a series of calibration curve solutions of 0.0005 IU / ml, 0.001 IU / ml, 0.005 IU / ml, 0.01 IU / ml, 0.02 IU / ml, 0.05 IU / ml and 0.1 IU / ml, and 6 series of calibration curve solutions were prepared in parallel. The blank matrix and the 6 series of calibration curve solutions were injected into the SPR detector (the chip used was obtained in Example 1) respectively. The results showed that the concentrations of the 6 series of calibration curve solutions were in the range of 0.0005~0.1 IU / ml, and the linear relationship was good, r>0.999; according to the data requirements (accuracy and precision were within ±25% of the labeled value), the lower limit of quantification (LLOQ) was confirmed to be 0.001 IU / ml, and the upper limit of quantification (ULOQ) was 0.1 IU / ml. The results are shown in Tables 2 and 3.

[0060] Table 2 Recovery results at each concentration point of the calibration curve

[0061] Table 3 Linearity results

[0062] (3) Accuracy and precision The tetanus human immunoglobulin national standard with a concentration of 0.001 IU / ml, 0.002 IU / ml, 0.016 IU / ml, 0.08 IU / ml and 0.1 IU / ml was prepared with a blank matrix, which was used as LLOQ, low concentration quality control, medium concentration quality control, high concentration quality control and ULOQ, respectively. Six portions of each concentration were prepared in parallel, and six series of calibration curve solutions were prepared (the series of calibration curve solutions were prepared by diluting the tetanus human immunoglobulin national standard with a blank matrix to a series of calibration curve solutions of 0.001 IU / ml, 0.005 IU / ml, 0.01 IU / ml, 0.02 IU / ml, 0.05 IU / ml and 0.1 IU / ml), and the blank matrix, six series of calibration curve solutions and six quality control solutions were injected into the SPR detector (the chip used was obtained in Example 1), respectively. Results: The accuracy of LLOQ and ULOQ in the six quality control solutions was within ±25% of the labeled value, and the accuracy of the low, medium and high concentration quality control solutions was within ±20% of the labeled value; the precision of the quality control at each concentration level was less than 20%. The results are shown in Table 4.

[0063] Table 4 Accuracy and precision results

[0064] (4) Dilution linearity A tetanus human immunoglobulin national standard with a concentration of 0.2 IU / ml was prepared with a blank matrix as a superlinear quality control product, and 6 portions were prepared in parallel. The 6 portions of superlinear quality control products were each diluted 10 times with the blank matrix to obtain 6 diluted quality control products with a concentration of 0.02 IU / ml. The blank matrix, a series of calibration curve solutions (the series of calibration curve solutions are the tetanus human immunoglobulin national standard diluted with the blank matrix to a series of calibration curve solutions of 0.001 IU / ml, 0.005 IU / ml, 0.01 IU / ml, 0.02 IU / ml, 0.05 IU / ml and 0.1 IU / ml) and 6 portions of diluted quality control products were respectively injected into the SPR detector (the chip used was obtained in Example 1). Results: The calculated concentrations of each diluted control were 103.9%, 97.26%, 101.1%, 98.21%, 98.21% and 94.41% of the labeled value, respectively; the precision was 3.31%.

[0065] (5) Parallelism A tetanus human immunoglobulin national standard with a concentration of 0.2 IU / ml was prepared using a blank matrix as a superlinear quality control product. The superlinear quality control product was diluted 4 times, 8 times and 16 times with the blank matrix to obtain low, medium and high concentration diluted quality control products with concentrations of 0.05 IU / ml, 0.025 IU / ml and 0.0125 IU / ml. The blank matrix, a series of calibration curve solutions (the series of calibration curve solutions are obtained by diluting the tetanus human immunoglobulin national standard with a blank matrix to a series of calibration curve solutions of 0.001 IU / ml, 0.005 IU / ml, 0.01 IU / ml, 0.02 IU / ml, 0.05 IU / ml and 0.1 IU / ml) and the diluted quality control product were injected into the SPR detector (the chip used was obtained in Example 1), respectively. Results: The calculated concentrations of low, medium and high concentration diluted quality control products were 97.14%, 95.05% and 98.95% of the labeled values, respectively; the precision was 2.01%.

[0066] (6) Stability The tetanus human immunoglobulin national standard with a concentration of 0.005 IU / ml and 0.08 IU / ml was prepared with a blank matrix as a low-concentration stability quality control and a high-concentration stability quality control, respectively. Eleven portions of each were prepared and placed at room temperature for 0h, 4h, 8h and 24h, at 4℃ for 4h, 8h and 24h, and at -20℃ for 24h and 48h. One portion of each low-concentration stability quality control and high-concentration stability quality control (the volume was enough for two determinations) was placed at -20℃ freeze-thaw conditions for 24h and 48h, and the blank matrix, a series of calibration curve solutions (the series of calibration curve solutions were prepared by diluting the tetanus human immunoglobulin national standard with a blank matrix to 0.001 IU / ml, 0.005 IU / ml, 0.01 IU / ml, 0.02 IU / ml, 0.05 IU / ml and 0.1 IU / ml series calibration curve solutions) and stability quality control products placed under various conditions were injected into the SPR detector (the chip used was obtained in Example 1). Results: The calculated concentrations of the stability quality control products placed under each condition were within ±20% of the labeled value; the precision was less than 20%, and the results are shown in Table 5.

[0067] Table 5 Stability results

[0068] Example 9 Real serum sample assay (1) Solution preparation: Blank matrix: Tetanus antibody negative serum was diluted 10 times with interaction buffer (1.0× PBS-P+) and filtered with a 0.2 μm filter membrane; Serial calibration curve solutions: dilute the tetanus human immunoglobulin national standard with blank matrix to prepare a series of calibration curve solutions of 0.001 IU / ml, 0.005 IU / ml, 0.01 IU / ml, 0.02 IU / ml, 0.05 IU / ml and 0.1 IU / ml; Test solution: Dilute the real serum sample infected with tetanus antibody 10 times with interaction buffer (1.0×PBS-P+), centrifuge at 12000r / min for 5min, and take the supernatant.

[0069] (2) Sample determination: first, a blank matrix was injected into a surface plasmon resonance instrument Biacore T200 (SPR detector), and the blank matrix was allowed to flow through the surface of the chip obtained in Example 1; a 0.001 IU / ml calibration curve solution was injected into a surface plasmon resonance instrument Biacore T200 (SPR detector), and the calibration curve solution was allowed to flow through the surface of the chip obtained in Example 1 to obtain the difference between the response signal of the 0.001 IU / ml calibration curve solution and the response signal of the blank matrix; the reaction buffer was used for dissociation for 100 s at a flow rate of 30 μl / min; then, a regeneration solution of glycine hydrochloride (Glycine pH 1.5) with a pH of 1.5 was injected into the chip at a flow rate of 30 μl / min for 120 s, and the chip was stabilized for 80 s after cleaning.

[0070] Inject a blank matrix into a surface plasmon resonance instrument Biacore T200 (SPR detector), and allow the blank matrix to flow through the surface of the chip obtained in Example 1; inject a 0.005 IU / ml calibration curve solution into a surface plasmon resonance instrument Biacore T200 (SPR detector), and allow the calibration curve solution to flow through the surface of the chip obtained in Example 1 to obtain the difference between the response signal of the 0.005 IU / ml calibration curve solution and the response signal of the blank matrix; dissociate with an interaction buffer for 100 s at a flow rate of 30 μl / min; then inject a regeneration solution of glycine hydrochloride (Glycine pH1.5) with a flow rate of 30 μl / min into the chip for 120 s, and stabilize for 80 s after the cleaning is completed.

[0071] Inject a blank matrix into a surface plasmon resonance instrument Biacore T200 (SPR detector), and allow the blank matrix to flow through the surface of the chip obtained in Example 1; inject a 0.01 IU / ml calibration curve solution into a surface plasmon resonance instrument Biacore T200 (SPR detector), and allow the calibration curve solution to flow through the surface of the chip obtained in Example 1 to obtain the difference between the response signal of the 0.01 IU / ml calibration curve solution and the response signal of the blank matrix; dissociate with an interaction buffer for 100 s at a flow rate of 30 μl / min; then inject a regeneration solution of glycine hydrochloride (Glycine pH 1.5) with a flow rate of 30 μl / min into the chip for 120 s, and stabilize for 80 s after the cleaning is completed.

[0072] Inject a blank matrix into a surface plasmon resonance instrument Biacore T200 (SPR detector), and allow the blank matrix to flow through the surface of the chip obtained in Example 1; inject a 0.02 IU / ml calibration curve solution into a surface plasmon resonance instrument Biacore T200 (SPR detector), and allow the calibration curve solution to flow through the surface of the chip obtained in Example 1 to obtain the difference between the response signal of the 0.02 IU / ml calibration curve solution and the response signal of the blank matrix; dissociate with an interaction buffer for 100 s at a flow rate of 30 μl / min; then inject a regeneration solution of glycine hydrochloride (Glycine pH 1.5) with a flow rate of 30 μl / min for 120 s, and stabilize for 80 s after cleaning.

[0073] Inject a blank matrix into a surface plasmon resonance instrument Biacore T200 (SPR detector), and allow the blank matrix to flow through the surface of the chip obtained in Example 1; inject a 0.05 IU / ml calibration curve solution into a surface plasmon resonance instrument Biacore T200 (SPR detector), and allow the calibration curve solution to flow through the surface of the chip obtained in Example 1 to obtain the difference between the response signal of the 0.05 IU / ml calibration curve solution and the response signal of the blank matrix; dissociate with an interaction buffer for 100 s at a flow rate of 30 μl / min; then inject a regeneration solution of glycine hydrochloride (Glycine pH 1.5) with a flow rate of 30 μl / min for 120 s, and stabilize for 80 s after cleaning.

[0074] A blank matrix was injected into a surface plasmon resonance instrument Biacore T200 (SPR detector), and the blank matrix was allowed to flow over the surface of the chip obtained in Example 1. A 0.1 IU / ml calibration curve solution was injected into a surface plasmon resonance instrument Biacore T200 (SPR detector), and the calibration curve solution was allowed to flow over the surface of the chip obtained in Example 1 to obtain the difference between the response signal of the 0.1 IU / ml calibration curve solution and the response signal of the blank matrix.

[0075] (3) Prepare a standard curve with the concentration of the series of calibration curve solutions as the horizontal axis and the difference in response signals of the calibration curve solutions with different concentrations as the vertical axis.

[0076] (4) After the standard curve is prepared, dissociate with the interaction buffer for 100 seconds at a flow rate of 30μl / min; then inject the regeneration solution of glycine hydrochloride (Glycine pH1.5) at a flow rate of 30μl / min for 120 seconds. After cleaning, stabilize for 80 seconds, then flow the blank matrix through the surface of the chip, and then flow the test solution through the surface of the chip to obtain the response signal 2; the response signal 2 = the response signal of the test solution - the response signal of the blank matrix; substitute the response signal 2 into the standard curve prepared in step (3) to obtain the tetanus antibody concentration in the test solution, and then multiply it by 10 times the dilution factor to obtain the tetanus antibody concentration in the real serum sample. The results are as follows Figure 8 As shown, the concentration of tetanus antibody in this real serum sample is 0.53 IU / ml.

[0077] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A chip for detecting tetanus antibody concentration in serum, characterized in that: The chip is a surface plasmon resonance chip coupled with tetanus toxoid, and the coupling level of the tetanus toxoid is 1000RU~10000RU.

2. The method for preparing the chip according to claim 1, characterized in that: The method comprises the following steps: first injecting EDC / NHS onto a surface plasma resonance chip fixed with carboxymethyl dextran; then injecting a tetanus toxoid solution to make the coupling level of the tetanus toxoid be 1000RU-10000RU; sealing to obtain the chip; the concentration of the tetanus toxoid in the tetanus toxoid solution is 24μg / ml-60μg / ml; and the pH value of the tetanus toxoid solution is 4.0-5.

0.

3. The preparation method according to claim 2, characterized in that: The flow rate of the tetanus toxoid solution was 5 μl / min and the injection time was 20 min.

4. Use of the chip according to claim 1 or the chip prepared by any one of the preparation methods of claims 2 to 3 in preparing a product for detecting tetanus antibody concentration in serum.

5. A kit for detecting tetanus antibody concentration in serum, characterized in that: The kit comprises the chip according to claim 1 or a chip prepared by any one of the preparation methods of claims 2 to 3.

6. The kit according to claim 5, characterized in that The test kit also includes a blank matrix, a tetanus human immunoglobulin standard and a regeneration solution; the blank matrix is ​​tetanus antibody negative serum.

7. A method for detecting tetanus antibody concentration in serum, characterized in that: The steps include: First, a blank matrix is ​​passed through the surface of the chip according to claim 1, and then different concentrations of tetanus human immunoglobulin standard solutions are passed through the surface of the chip to obtain response signals 1 of different concentrations; the response signal 1 = the response signal of the tetanus human immunoglobulin standard solution - the response signal of the blank matrix; A standard curve was prepared with the concentration of tetanus human immunoglobulin standard solution as the abscissa and the response signal 1 of different concentrations as the ordinate; After the chip is cleaned with a regeneration solution, a blank matrix is ​​first passed through the surface of the chip, and then a serum sample to be tested is passed through the surface of the chip to obtain a response signal 2; the response signal 2 = the response signal of the serum sample to be tested - the response signal of the blank matrix; The response signal 2 is substituted into the standard curve to calculate the tetanus antibody concentration in the serum sample to be tested.

8. The method according to claim 7, characterized in that The standard curve is y=ax+b, R 2 >0.99, the x is the tetanus antibody concentration, the y is the response signal, the a>500, and the absolute value of the b is <3.

9. The method according to claim 7, characterized in that: The regeneration solution is a glycine solution, and the pH of the glycine solution is 1.

5.

10. The method according to claim 7, characterized in that The steps of regeneration solution cleaning are as follows: injecting the regeneration solution into the chip at a flow rate of 30 μl / min, the regeneration time is 120 s, and the regeneration is stabilized for 80 s.

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