Tetanus antibody titer determination method

Abstract

The invention relates to the technical field of biological medicine detection, and discloses a tetanus antibody titer determination method, which comprises: 1, diluting a standard substance; 2, diluting a sample to be detected; step 3, adding a sample and incubating, adding the R1 reagent and the standard substance / sample, incubating, and measuring an absorbance value A1; 4, after incubation is completed, a reagent R2 is added for incubation, and the absorbance value A2 is measured; and 5, calculating an absorbance difference value A = A2-A1, sorting the absorbance value of the standard substance, fitting an absorbance-titer function by using statistical software, and calculating the titer content of the tetanus antibody according to the absorbance value of the sample and the fitting function. According to the invention, the tetanus human immune globulin national standard product is used as a calibration product to be calibrated on a biochemical analyzer, and then the tetanus antibody titer of a test sample is determined by using the tetanus antibody detection reagent solution, so that the titer of the test sample can be rapidly and accurately determined, and the cost is far lower than that of a mouse method.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical detection, and in particular to a method for determining the titer of tetanus antibodies. Background Art

[0002] Tetanus is a specific infection caused by Clostridium tetani invading human wounds. It is a fatal infectious disease. Its highly toxic spasmodic toxin can block the inhibitory neurotransmitters of the central nervous system, causing muscle rigidity and typical systemic spasms. Antibody titer is an important indicator in the fields of disease diagnosis, specific immunoglobulin preparation and vaccine evaluation, and plays a very important role. By detecting the tetanus antibody titer, the immune protection level of individuals after vaccination can be evaluated and the effectiveness of the vaccine can be judged. Determining the tetanus antibody titer can help understand the immune status of individuals after natural infection and evaluate their resistance to reinfection.

[0003] Current methods for testing tetanus antibody titers include:

[0004] Neutralization test method: Diluted serum is mixed with tetanus toxin, then added to sensitive mice or cell lines, the lethal effect or cytopathic effect (CPE) of the toxin is observed, and the titer of neutralizing antibodies is calculated. However, this method requires a long incubation time, and it usually takes several days to get the results. The experimental results are affected by many factors, such as the sensitivity of the mice, the state of the cell line, etc., which may lead to inconsistent results.

[0005] Enzyme-linked immunosorbent assay (ELISA): This method indirectly reflects the titer of the antibody by detecting the binding ability of specific tetanus antibodies in serum to tetanus antigens fixed on a solid phase carrier. However, the kit cost of this method is high, and there is a certain probability of false positives or false negatives, which requires further confirmation. In addition, ELISA has many operating steps, requires precise sample addition and washing steps, and is prone to operational errors.

[0006] Turbidimetry: The titer of the antibody is evaluated by measuring the turbidity change of the immune complex formed after the antibody binds to the antigen. However, this method has a relatively low sensitivity and may not be able to detect low-titer antibodies; and the specificity of the turbidimetry is relatively poor and may be interfered by nonspecific immune complexes.

[0007] Therefore, there is an urgent need to develop a method that can quickly and accurately detect tetanus antibody titer. Summary of the invention

[0008] The present invention aims to provide a method for determining the tetanus antibody titer, so as to achieve rapid and accurate detection of the tetanus antibody titer.

[0009] To achieve the above object, the present invention adopts the following technical scheme: a method for determining the titer of tetanus antibodies, comprising the following steps:

[0010] Step 1: dilution of standard;

[0011] Step 2: diluting the sample to be tested;

[0012] Step 3: Adding samples and incubating: adding R1 reagent and sample / standard, incubating, and measuring the absorbance value A1;

[0013] Step 4: After the incubation is completed, add the R2 reagent and incubate, and measure the absorbance value A2;

[0014] Step 5: Calculate the absorbance difference △A=A2-A1, sort out the absorbance values ​​of the standard, use statistical software to fit the absorbance-potency function, and calculate the tetanus antibody content based on the fitting function.

[0015] Preferably, as an improvement, in step three, the absorbance value A1 is measured at a wavelength of 570nm / 700nm.

[0016] Preferably, as an improvement, in step three, the incubation condition is incubating in a reaction plate at 37° C. for 5 minutes.

[0017] Preferably, as an improvement, in step 4, reagent R1 and reagent R2 are placed at room temperature for 30 minutes before use.

[0018] In this technical solution, R1 and R2 are two reagents in the tetanus immune plasma potency test kit, including a diluent (composed of a buffer solution, a surfactant, a turbidity promoter, and a preservative) and a working solution (composed of a buffer solution, latex particles coated with a tetanus vaccine toxoid stock solution, unrelated proteins, and a preservative).

[0019] Preferably, as an improvement, in step 4, the absorbance value A2 is measured at a wavelength of 570nm / 700nm.

[0020] Preferably, as an improvement, in step 4, the incubation condition is incubating in a reaction plate at 37° C. for 5 minutes.

[0021] Preferably, as an improvement, in step five, the highest dilution causing a significant turbidity change is the tetanus antibody titer.

[0022] The principle and advantage of this scheme are: in practical application, in view of the problems of poor repeatability and accuracy in the prior art when detecting the tetanus antibody titer, in this technical scheme, when detecting the tetanus antibody titer, based on turbidimetry, its principle is: based on the surface cross-linked monoclonal antigens on the polymer latex particles, it is allowed to specifically bind to the antibody to be tested in a buffer containing a polymerization promoter to form an immune complex. The particle size and number of these complexes will vary with the different antibody titers. The antibody titer is quantitatively analyzed by using a fully automatic biochemical analyzer to detect the turbidity changes caused by the immune complexes formed in the reaction system. The increase in turbidity is proportional to the amount of immune complexes generated, and the amount of immune complexes generated is related to the concentration and titer of the antibody. By establishing a standard curve, the turbidity value of the sample to be tested is compared with a standard of known titer, so as to determine the titer of tetanus antibodies in the sample to be tested.

[0023] Tetanus antigens specifically bind to tetanus antibodies in serum to form immune complexes. The formation of immune complexes will cause the turbidity of the solution to increase, and the antibody titer can be assessed by measuring the change in turbidity.

[0024] During the technical research and development period, this technical solution abandoned the unclear calibration and calculation methods of the biochemical analyzer, broke the existing legal methods and the existing calibration and calculation methods of the instrument, creatively used the tetanus human immunoglobulin national standard as a calibrator on the biochemical analyzer, and then used the tetanus antibody detection reagent solution to determine the tetanus antibody titer of the test sample. When calculating the results, all absorbance values ​​were read, and curve fitting and calculation were completed in EXCEL, making the detection transparent. The detection time is about 30 minutes, the cost of a single batch is about 500 yuan / batch, and the cost of multiple batches is about 300 yuan / batch. It can achieve rapid and accurate determination of the titer of the test sample, and the cost is much lower than the mouse method. (In the tetanus antibody titer determination experiment, the mouse method specified in the pharmacopoeia is currently used. The consumables required for the experiment include national standards, toxins, mice, feed, bedding, sterilized water, etc. The cost of testing a batch of test samples is about 2,000 yuan / batch, and the cost is 1,800 yuan / batch when multiple batches are carried out simultaneously. It also takes 5 days to complete the test.) In this process, the determination of the dilution multiples of the standard and the sample to be tested is one of the key points of this technical solution: the original method (the concentration of the standard is: 0IU / ml, 2.5IU / ml, 5IU / ml, 10IU / ml, 20IU / ml, 30IU / ml, the detection range is 0-30IU / ml, and the correlation coefficient r>0.99. Detection method: immunoturbidimetry. Main wavelength: 578 nm, secondary wavelength: 700nm, calibration method: four parameters. Reaction direction: forward. R1 reagent 150μl; R2 reagent 50μl. Determination method (two-point endpoint method): 150μl diluent is added to 2μl sample, react at 37℃ for 5 minutes, read the point to measure the absorbance A1, then add 50μl working solution, react at 37℃ for 5 minutes, read the point again to measure the absorbance A2, calculate the absorbance difference, and calculate the test result according to the tetanus standard curve) When detecting high-titer samples, the repeatability and accuracy of the test results are poor due to the large dilution multiple, so it is considered to use the calibrated sample to make a curve, fit a curve suitable for detecting high-concentration samples, so as to reduce the dilution multiple of the sample, but this method is limited by the preservation of the sample and the reliability of the results, so it is abandoned.

[0025] In summary: The beneficial effects of this technical solution are: this technical solution uses the national standard of tetanus human immunoglobulin as a calibration product to make a standard curve, has an accurate and reliable data source, and the test results are stable and reliable. This method saves costs while complying with animal welfare ethics and reducing the biosafety risks of personnel using tetanus toxin. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the standard curve of tetanus antibody titer test in the embodiment of the present invention. DETAILED DESCRIPTION

[0027] The following is further described in detail through specific implementations, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following implementations are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; the materials, reagents, etc. used can all be obtained from commercial channels.

[0028] A method for determining tetanus antibody titer comprises the following steps:

[0029] Step 1: Establish a standard curve: Use the national standard of human tetanus immunoglobulin as the calibration product;

[0030] Step 2: dilute the sample to be tested to an appropriate concentration for standby use;

[0031] Step 3: Pipette 2 μl of the diluted sample into a reaction cup, add 150 μl of R1 reagent, incubate in a 37°C reaction plate for 5 min, and measure the absorbance value A1 at a wavelength of 570 nm / 700 nm.

[0032] Step 4: After the incubation time is completed, the biochemical analyzer automatically aspirates and adds 50 μl of R2 reagent, incubates in a 37°C reaction plate for 5 minutes, and measures the absorbance value A2 at a wavelength of 570 nm / 700 nm.

[0033] Step 5: The biochemical analyzer automatically calculates the absorbance difference △A=A2-A1, and the instrument automatically converts the tetanus antibody content according to the generated standard curve.

[0034] illustrate:

[0035] Name: Fully automatic biochemical analyzer Manufacturer: Shenzhen Mindray Bio-Medical Electronics Co., Ltd.

[0036] Instrument model: BS-2200 No.: HLYX-SY-009

[0037] Key Reagents:

[0038] Tetanus human immunoglobulin national standard (purchased from China Food and Drug Inspection Institute);

[0039] Kit (tetanus immune plasma titer test reagent, purchased from Shandong Dijun Biotechnology Co., Ltd.);

[0040] R1 and R2 are two reagents in the tetanus immune plasma titer test kit: the kit was purchased from Shandong Dijun Biotechnology Co., Ltd., stored at 2-8°C, and is prohibited from freezing. The validity period is 12 months, and it should be placed for 30 minutes before use to balance to the temperature. It includes diluent (consisting of buffer, surfactant, turbidity promoter, and preservative) and working solution (consisting of buffer, latex particles coated with tetanus vaccine toxoid stock solution, unrelated proteins, and preservatives).

[0041] Experimental Example 1

[0042] Experimental methods:

[0043] Step 1, establish a standard curve: dilute 1 bottle of tetanus human immunoglobulin national standard step by step to obtain 18 IU / ml, 9 IU / ml, 4.5 IU / ml, 2.25 IU / ml, 1.125 IU / ml working standard solutions for use;

[0044] Step 2: Take 2 ml of a batch of tetanus human immunoglobulin stock solution into a 50 ml volumetric flask, dilute to volume with purified water, shake well, and set aside;

[0045] Step 3. Take the prepared working standard solution and place it on the sample rack of the automatic biochemical analyzer. Use the sample needle to draw 3μl of the blank and working standard solutions into the reaction cup, add 150μl of R1 reagent, incubate in a 37℃ reaction plate for 5 minutes, and measure the absorbance value A1. After the incubation time is up, add 50μl of R2 reagent, incubate in a 37℃ reaction plate for 5 minutes, measure the absorbance value A2, and the instrument automatically calculates the absorbance difference △A=A2-A1. At this time, record the absorbance value corresponding to the concentration of each working standard solution;

[0046] Step 4. Take the prepared sample solution to be tested and place it on the sample rack of the fully automatic biochemical analyzer. Pour 3μl of the sample blank and sample solution into the reaction cup, add 150μl of R1 reagent, incubate in a 37℃ reaction plate for 5 minutes, and measure the absorbance value A1. After the incubation time is up, add 50μl of R2 reagent, incubate in a 37℃ reaction plate for 5 minutes, measure the absorbance value A2, and the instrument automatically calculates the absorbance difference △A=A2-A1. At this time, record the absorbance value of the sample solution;

[0047] Step 4: Use statistical software to fit the potency-absorbance value function of the standard, and substitute the absorbance value of the sample solution into the function to obtain the tetanus antibody titer.

[0048] Experimental results:

[0049] Working Standards:

[0050] Table 1

[0051] Potency 0 1.125 2.25 4.5 9 18 Absorbance value 0.1294 0.1331 0.1369 0.1444 0.1619 0.1960

[0052] Sample absorbance: 0.1448, the obtained standard curve is as follows Figure 1 As shown, R 2 =0.99988;

[0053] Calculation result: 46.94974*((0.12943-0.1912) / (0.1912-0.40888))^(1 / 1.22877)=16.84IU / ml

[0054] Original solution potency = 16.84 IU / ml*25 = 215.1 IU / ml

[0055] Experimental Example 2 Comparison of the detection effects of mouse method and immunoturbidimetry

[0056] The method of the present invention (immunoturbidimetry).

[0057] Comparative method (mouse method): refer to General Chapter 3508 of Part IV of the Chinese Pharmacopoeia, or refer to the document "Standard Operating Procedure for Tetanus Antibody Titer Determination".

[0058] The test results are shown in Table 2: The error refers to the error between the immunoturbidimetric method of the present technical solution and the mouse method, and the calculation method is: (immunoturbidimetric method test result B-mouse method test result A) / mouse method test result A. The results show that the error between the test method of the present technical solution and the mouse method is between -0.56% and 5.42%.

[0059] Table 2

[0060]

[0061] The present invention optimizes the parameters based on the immunoturbidimetry method:

[0062] Original parameters: The concentration of the standard is: 0IU / ml, 2.5IU / ml, 5IU / ml, 10IU / ml, 20IU / ml, 30IU / ml, the detection range is 0-30IU / ml, and the correlation coefficient r>0.99. Detection method: immunoturbidimetry. Main wavelength: 578nm, secondary wavelength: 700nm, calibration method: four parameters. Reaction direction: forward. R1 reagent 150μl; R2 reagent 50μl. Determination method (two-point endpoint method): 150μl diluent is added to 2μl sample, react at 37℃ for 5 minutes, read the point to measure the absorbance A1, then add 50μl working solution, react at 37℃ for 5 minutes, read the point again to measure the absorbance A2, calculate the absorbance difference, and calculate the test result according to the tetanus standard curve.

[0063] Optimized parameters: The concentration of the standard is: 0IU / ml, 1.125IU / ml, 2.25IU / ml, 4.5IU / ml, 9IU / ml, 18IU / ml, the detection range is 0-18IU / ml, and the correlation coefficient r>0.999. Detection method: immunoturbidimetry. Main wavelength: 578nm, secondary wavelength: 700nm, calibration method: five parameters. Reaction direction: forward. R1 reagent 150μl; R2 reagent 50μl. Determination method (two-point endpoint method): 150μl diluent is added to 3μl sample, react at 37℃ for 5 minutes, read the point to measure the absorbance A1, then add 50μl working solution, react at 37℃ for 5 minutes, read the point again to measure the absorbance A2, calculate the absorbance difference, and calculate the test result according to the tetanus standard curve.

[0064] The detection effect before and after parameter optimization was tested and evaluated. The specific experimental design and test results are shown in Table 3-5.

[0065] Table 3 Comparison of precision before and after parameter optimization (dosage addition)

[0066]

[0067]

[0068] Table 4 Linear comparison before and after parameter optimization (fitting function)

[0069]

[0070] Table 5 Comparison of accuracy before and after parameter optimization (fitting function) (standard diluted to 9 IU / ml and added to the appropriate sample matrix)

[0071]

[0072] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A method for determining tetanus antibody titer, characterized in that: The steps include: Step 1: dilution of standard; Step 2: diluting the sample to be tested; Step 3: Adding samples and incubating: adding R1 reagent and sample / standard, incubating, and measuring the absorbance value A1; Step 4: After the incubation is completed, add the R2 reagent and incubate, and measure the absorbance value A2; Step 5: Calculate the absorbance difference △A=A2-A1, sort out the absorbance values ​​of the standard, use statistical software to fit the absorbance-potency function, and calculate the tetanus antibody content based on the fitting function.

2. A method for determining tetanus antibody titer according to claim 1, characterized in that: In step 3, the absorbance value A1 is measured at a wavelength of 570nm / 700nm.

3. A method for determining tetanus antibody titer according to claim 2, characterized in that: In step 3, the incubation condition is incubating in a reaction plate at 37° C. for 5 minutes.

4. A method for determining tetanus antibody titer according to claim 3, characterized in that: Reagent R1 and reagent R2 were placed at room temperature for 30 min before use.

5. A method for determining tetanus antibody titer according to claim 4, characterized in that: In step 4, the absorbance value A2 is measured at a wavelength of 570nm / 700nm.

6. A method for determining tetanus antibody titer according to claim 5, characterized in that: In step 4, the incubation condition is incubating in a reaction plate at 37° C. for 5 minutes.

7. A method for determining tetanus antibody titer according to claim 6, characterized in that: In step 5, the highest dilution that causes a significant turbidity change is the tetanus antibody titer.

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