Automatic micro-column for Trust test and method for treating irregular antibody titer in serum
By designing a Trust test automated microcolumn, using specific volume ratio mixing of glycerol and low-ion media, and combining QR code technology to achieve the interconnection of sample information and patient information, it solves the random error problem caused by manual operation of traditional Trust tests, and significantly improves the sensitivity and accuracy of the detection results.
Patent Information
- Application Number
- CN202411534442.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-31
- Publication Date
- 2025-03-11
AI Technical Summary
The traditional Trust test was manually operated throughout the whole process, and the results were interpreted with the operator's naked eye recognition, resulting in the occurrence of random errors and reducing the accuracy and sensitivity of irregular antibodies.
A Trust test automated microcolumn is designed to form a microcolumn by mixing glycerol and low-ion media in a specific volume ratio, combining QR code technology to achieve the interconnection of sample information and patient information, achieving fully automated operations, and reducing the interference of human factors on the results.
The sensitivity of the test results is improved, the probability of false negatives or false positives is reduced, and the accuracy of the test results is enhanced. Compared with the traditional paper sheet method, the sensitivity is 8 times higher.
Smart Images
Figure CN119595888A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biomedicine, and in particular to a Trust test automated microcolumn and a method for processing irregular antibody titer in serum. Background Art
[0002] The treatment of early sexual syphilis is extremely simple. After the diagnosis is confirmed, good therapeutic effects can be achieved by using penicillin (cephalosporin antibiotics can be used for those who are allergic to penicillin).
[0003] In actual clinical diagnosis and treatment, whether to use antibiotics for treatment and the evaluation of the effectiveness of antibiotic treatment mainly rely on the results of traditional Trust or RPR tests to analyze the nonspecific antibody titer in the patient. Currently, the Trust test is performed manually and the results are interpreted by the operator's naked eye. As a result of different operators and different operating times, random errors are inevitable, which reduces the accuracy and sensitivity of the reagent for irregular antibodies.
[0004] In view of this, the present invention is proposed. Summary of the invention
[0005] The first purpose of the present invention is to provide a Trust test automated microcolumn, which has simple components and can realize the titer detection of irregular antibodies, effectively improving the sensitivity of detection. When a two-dimensional code of size (1cm*1cm) is designed at the tail of the card on the basis of the automated microcolumn of the present invention, the card and the patient sample information can be interconnected one-to-one, thereby realizing the full automation of the test method and reducing the interference of human factors and random errors on the results.
[0006] The second object of the present invention is to provide a method for processing irregular antibody titers in serum using the Trust test automated microcolumn, which can effectively improve the sensitivity of the detection results.
[0007] The third object of the present invention is to provide a method for using a Trust test automation microcolumn and a method for processing irregular antibody titer in serum using the Trust test automation microcolumn in a syphilis detection kit.
[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted: The invention provides a Trust test automation microcolumn, which is prepared by mixing glycerol and a low-ion medium in a volume ratio of (1:5) to (1:4).
[0009] When Treponema pallidum destroys the body's tissues, it releases a cardiolipin antigen, which can stimulate the body to produce corresponding antibodies, namely reactins. The irregular antibodies produced by Treponema pallidum after invading the human immune response are negatively charged in neutral solutions, and cardiolipin is also negatively charged. Low-ion media are used to reduce the repulsion of electric field forces between charges of the same kind in the entire reaction system; at the same time, low-ion media can be used as a good solvent to dissolve all glycerol and prevent glycerol from stratification. Glycerol can play a good separation role and can precipitate the colored antigen-antibody complex. Common dextran dry powder is easy to be tested for making gels or other gels. When the concentration is too high, it will crack and cannot be loaded into the column, let alone test its molecular sieve effect. Because the molecular weight of the separated antigen-antibody complex is too large, low-speed centrifugation cannot separate it to the bottom of the microcolumn, so the purpose of detection cannot be achieved; and when raw materials such as alumina are used, the sensitivity of the kit will be reduced, resulting in a high false positive rate.
[0010] At this volume ratio, the low-ion medium can be used as a solvent to dissolve all the glycerol well, while ensuring the separation effect of glycerol. When the glycerol content is low, it is difficult to ensure the separation of the antigen-antibody complex, and when the glycerol is excessive, it will lead to a low content of low-ion medium, thereby increasing the repulsion effect of like charges in the reaction system, resulting in false positives.
[0011] Preferably, as a further specific embodiment, the low-ion medium is any one or more of 5wt% glucose or 0.2wt% disodium ethylenediaminetetraacetate.
[0012] In the present invention, the use of the above-mentioned low-ion medium can better balance the electric field force in the reaction system, reduce the repulsive effect of like charges, and at the same time promote the reaction between irregular antibodies and cardiolipin in serum, thereby improving the sensitivity of the reaction.
[0013] The present invention also provides a method for processing samples using the Trust test automated microcolumn, comprising the following steps: Take a sample and mix it with 0.9wt% saline in a volume ratio of 1:2 n Dilute by multiple times, where n≥1, water bath, centrifuge, and obtain the original dilution solution; The original dilution solution and the Trust test automated microcolumn are mixed and stirred to obtain the product.
[0014] In the present invention, the selected sample is an isolated serum or plasma sample, which is treated with physiological saline to ensure the antibody activity in the blood sample. nThe gradient dilution can, on the one hand, provide a buffer system for the antibody to facilitate the binding of the antigen and antibody; on the other hand, it can protect the antibody, keep the antibody stable and prevent the antibody titer from decreasing; finally, the gradient dilution can show the reaction results more clearly and reduce the probability of false positives or false negatives.
[0015] Preferably, as a further specific implementation, n=5.
[0016] When the volume ratio of the sample to 0.9wt% saline is 1:32, the sample diluted in this way can form a stable reaction system, and the sensitivity of the diluted system is the highest.
[0017] Preferably, as a further specific embodiment, the temperature of the water bath is 35°C-40°C, and the time of the water bath is 9min-15min; Preferably, the water bath temperature is 37° C., and the water bath time is 10 min.
[0018] In the present invention, the reaction temperature and reaction time are limited. For the reaction of antigen and antibody, the reaction temperature cannot exceed its optimal temperature. High temperature will inactivate them, while low temperature cannot fully react. Therefore, 35°C-40°C is the most appropriate; 37°C is the optimal reaction temperature.
[0019] In this reaction system, the reaction time is set to 9min-15min. The reaction time should not be too long, as too long a reaction time will reduce the sensitivity of the reaction system. When the reaction time is too short, the antigen-antibody complex cannot fully combine and precipitate, resulting in inaccurate test results.
[0020] Preferably, as a further specific embodiment, the centrifugal speed of the centrifugation is 1100rpm / min-1500rpm / min, and the centrifugal time is 8min-12min; Preferably, the centrifugal speed is 10000 rpm / min, and the centrifugal time is 9 min.
[0021] Preferably, as a further specific embodiment, the stirring speed is 3000rpm / min-5000rpm / min; Preferably, the stirring speed is 3500 rpm / min.
[0022] In the reaction process of the present invention, the centrifugal speed and time need to be limited. The centrifugal speed should not be too high and the centrifugal time should not be too long. The reason is that a large pressure will be generated during the centrifugation process, which will destroy the structure of the antigen-antibody complex and affect the detection results. The centrifugal speed should not be too low and the time should not be too short. The reason is to prevent the subsequent inability to fully centrifuge and precipitate, which ultimately reduces the sensitivity of the reaction system.
[0023] Compared with the prior art, the present invention has the following beneficial effects: The automated microcolumn of the present invention can effectively improve the sensitivity of detection. Compared with the sensitivity of 1 / 8 of the traditional paper disc method, the present invention increases the sensitivity of binding with syphilis virus to 1 / 128, reducing the probability of false negative or false positive. At the same time, it can reduce the random error of experimental results caused by human observation factors during paper disc method detection, and improve the accuracy of detection results. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 : Sample test results using the paper disc method for sample 1; Figure 2 : Sample test results using the paper disc method for sample 2; Figure 3 : Experimental results after using Trust test automated microcolumn to process sample 1, sample 2, sample 3 and sample 4, where serial number 6 is the test result of sample 1, serial number 2 is the test result of sample 2, serial number 3 is the test result of sample 3, and serial number 7 is the test result of sample 4; Figure 4 : Sample 3 was tested using the paper disc method, and the negative control group 1 was finally obtained; Figure 5 : Sample 4 was tested using the paper strip method, and the negative control group 2 was finally obtained. DETAILED DESCRIPTION
[0025] The technical scheme of the present invention will be clearly and completely described below in conjunction with specific embodiments, but it will be appreciated by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work, all belong to the scope of protection of the present invention. If specific conditions are not indicated in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not indicated in the reagents or instruments used, they are all conventional products that can be purchased commercially.
[0026] Example 1 Take sample 1 and directly test it using the paper disc method. The test results are as follows: Figure 1As shown, the paper disc method showed that the sample was a strongly positive sample.
[0027] Take sample 1, dilute sample 1 with 0.9wt% normal saline at a volume ratio of 1:32, heat in a water bath to 37°C for 10 minutes, and obtain a stock solution dilution; The obtained diluted stock solution was centrifuged at a centrifugal speed of 1100 rpm / min for 9 minutes to obtain a complex; 2 mL of glycerol and 10 mL of 0.2 wt % disodium ethylenediaminetetraacetate (volume ratio of 1:5) were mixed to obtain a microcolumn, and the complex was added to the microcolumn and stirred at a stirring speed of 1100 rpm / min for 5 minutes to obtain a treated sample 1.
[0028] The processed sample 1 is tested, and the experimental results are as follows Figure 3 As shown in sequence number 6, this sample is a strongly positive sample.
[0029] Example 2 Take sample 2 and directly test it using the paper disc method. The test results are as follows: Figure 2 As shown, the test results showed that sample 2 was negative.
[0030] Take sample 2, dilute sample 2 with 0.9wt% normal saline at a volume ratio of 1:32, heat in a water bath to 37°C for 10 minutes, and obtain a stock solution dilution; The obtained diluted stock solution was centrifuged at a centrifugal speed of 10000 rpm / min for 9 minutes to obtain a complex; 2 mL of glycerol and 10 mL of 0.2 wt % disodium ethylenediaminetetraacetate (volume ratio of 1:5) were mixed to obtain a microcolumn, and the complex was added to the microcolumn and stirred at a stirring speed of 3500 rpm / min for 5 minutes to obtain a treated sample 2.
[0031] The processed sample 2 is tested, and the experimental results are as follows Figure 3 As shown in sequence number 2, this sample is a strongly positive sample.
[0032] The patient from whom sample 2 was derived was followed up and confirmed to be a positive patient.
[0033] Example 3 Samples 3 and 4 were directly tested using the paper disc method. The test results are as follows: Figure 4 , Figure 5 As shown, the test results showed that sample 3 and sample 4 were negative samples.
[0034] Sample 3 and sample 4 were taken respectively, and sample 3 and sample 4 were diluted with 0.9wt% normal saline at a volume ratio of 1:32, and heated to 37°C in a water bath for 10 minutes to obtain the original solution dilutions; The obtained diluted stock solution was centrifuged at a centrifugal speed of 10000 rpm / min for 9 minutes to obtain a complex; 2 mL of glycerol and 10 mL of 0.2 wt % disodium ethylenediaminetetraacetate (volume ratio of 1:5) were mixed to obtain a microcolumn. The complex was added to the microcolumn and stirred at a stirring speed of 3500 rpm / min for 5 minutes to obtain treated samples 3 and 4.
[0035] The processed samples 3 and 4 were tested, and the experimental results are as follows Figure 3 Serial number 3 and Figure 3 As shown in serial number 7, the above two samples are both negative samples.
[0036] By comparing the paper disc method in Examples 1 to 3 and the treatment using the microcolumn of the present invention, it is found that no false negative or false positive results will occur when the microcolumn of the present invention is used for detection, and the detection results are relatively accurate.
[0037] Example 4 The specific implementation method is the same as that of Example 1. Different dilution gradient processing methods are used to process the samples and their sensitivity is tested, as shown in Table 1: Table 1: Changes in sensitivity of samples and 0.9wt% saline at different dilution gradients
[0038] From the above experimental results, it can be seen that after gradient dilution, the closer the ratio of the two is to 1:32, the higher the sensitivity. The reason is that at a ratio close to 1:32, the reaction is more stable and a buffer system is formed that is conducive to antigen-antibody binding, thereby improving sensitivity.
[0039] Example 5 The specific implementation is the same as Example 1, except that 0.2wt% disodium ethylenediaminetetraacetate is replaced by 5wt% glucose solution, and the sensitivity is slightly decreased, but it can be ignored. It can be seen that 0.2wt% disodium ethylenediaminetetraacetate and 5wt% glucose solution have basically the same ability to balance the same charges in the reaction system.
[0040] Example 6 The specific implementation method is consistent with Example 1, and the sample processing method is changed, as shown in Table 2: Table 2: Changes in sensitivity due to different treatment methods
[0041] It can be seen from Example 6 that the sensitivity will be reduced when the sample processing conditions are changed. The main reason is that the change in the processing method will affect the reaction system, so that the highest sensitivity cannot be achieved. It can be seen from the experimental results that the reaction water bath temperature is set at 35°C-40°C, the water bath time is set at 9min-15min; the centrifugal speed is set at 5000rpm / min-12000rpm / min, the centrifugal time is set at 8min-12min; the stirring time is set at 3000rpm / min-5000rpm / min. The sensitivity is the highest and the technical effect of the present invention can be achieved.
[0042] Comparative Example 1 The specific implementation method is consistent with Example 1, except that the low-ion medium is not used.
[0043] Without using low-ionic media, the charges cannot be balanced, rendering the experimental results meaningless.
[0044] Comparative Example 2 The specific implementation method is consistent with Example 1, except that the volume ratio of glycerol to the low-ion medium is adjusted to: glycerol: 0.2wt% disodium ethylenediaminetetraacetic acid volume ratio = 2:1.
[0045] After the adjustment of the reaction system, glycerol cannot be dissolved in low-ion media, making the experimental results meaningless.
[0046] Comparative Example 3 The specific implementation method is consistent with Example 1, except that the volume ratio of glycerol to the low-ion medium is adjusted to: glycerol: 0.2wt% disodium ethylenediaminetetraacetic acid volume ratio = 8:1.
[0047] After the adjustment of the reaction system, the antigen-antibody complex cannot be precipitated, resulting in meaningless experimental results.
[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. A Trust test automated microcolumn, characterized in that: It is prepared by mixing glycerol and low-ion medium in a volume ratio of (1:5) - (1:4).
2. The Trust test automation microcolumn according to claim 1, characterized in that: The low-ion medium is any one or more of 5wt% glucose or 0.2wt% disodium ethylenediaminetetraacetate.
3. A method for treating irregular antibody titers in serum using a Trust test automated microcolumn as claimed in any one of claims 1 to 2, characterized in that: The steps include: Take a sample and mix it with 0.9wt% saline in a volume ratio of 1:2 n Dilute by multiple times, where n≥1, water bath, centrifuge, and obtain the original dilution solution; The original dilution solution and the Trust test automated microcolumn are mixed and stirred to obtain the product.
4. The method for treating irregular antibody titers in serum using Trust test automated microcolumns according to claim 3, characterized in that: Said n=5.
5. The method for treating irregular antibody titer in serum using Trust test automated microcolumn according to claim 3, characterized in that: The temperature of the water bath is 35°C-40°C, and the time of the water bath is 9min-15min; Preferably, the water bath temperature is 37° C., and the water bath time is 10 min.
6. The method for treating irregular antibody titer in serum using Trust test automated microcolumn according to claim 3, characterized in that: The centrifugal speed of the centrifugation is 1100rpm / min-1500rpm / min, and the centrifugal time is 8min-12min; Preferably, the centrifugal speed is 10000 rpm / min, and the centrifugal time is 9 min.
7. The method for treating irregular antibody titer in serum using Trust test automated microcolumn according to claim 3, characterized in that: The stirring speed is 3000rpm / min-5000rpm / min; Preferably, the stirring speed is 3500 rpm / min.
8. Use of the Trust test automation microcolumn as described in any one of claims 1 to 2 and the method for processing irregular antibody titer in serum using the Trust test automation microcolumn as described in claims 3 to 7 in a syphilis detection kit.