Varicella-zoster virus combined diagnosis detection kit

Abstract

The invention discloses a varicella-herpes zoster virus combined diagnosis detection kit, which relates to the technical field of biomedicine and clinical diagnosis, and comprises a 96-well plate pre-coated with recombinant herpes zoster virus glycoprotein, an HRP enzyme-labeled second antibody, a substrate solution, a stop solution, a washing solution, a sample diluent, a positive control and a negative control, according to the chicken pox-herpes zoster virus combined diagnosis and detection kit, the 96-well plate is coated with the recombinant herpes zoster virus glycoprotein, and I gM and I gG are subjected to combined detection, so that the infection condition of chicken pox-herpes zoster virus can be reflected more comprehensively; in the initial stage of virus infection, the positive result of the IgM prompts that a patient is possibly in an acute infection stage, and along with the progress of infection, the gradual increase of the IgG indicates that an organism has generated a long-term immune response, which is crucial for evaluating the immune state of the patient, predicting the disease development trend and formulating a subsequent treatment plan.

Description

Technical Field

[0001] The invention relates to biomedicine and clinical diagnosis technology, and in particular to a varicella-zoster virus combined diagnosis and detection kit. Background Art

[0002] Varicella-zoster virus (VZV) is a double-stranded DNA virus, also known as human herpesvirus type 3, which is mainly transmitted from person to person. Varicella-zoster virus is mainly transmitted through respiratory droplets and direct contact, and can also be transmitted indirectly through utensils. After the initial infection with varicella-zoster virus, the virus will lurk inside the dorsal root ganglia of the spinal cord or the sensory ganglia of the cranial nerves, evading the surveillance of the body's immune system. When the body's immunity decreases, the virus can be reactivated and reach the skin along the nerve axons, causing skin damage and blisters. At the same time, the nerves can also be damaged, causing obvious pain. This is a typical manifestation of shingles.

[0003] There are at least nine glycoproteins (gps) on the VZV capsid, which are concentrated in the viral envelope and the cell membrane of infected cells and are closely related to the pathogenicity and immunogenicity of the virus. The most important gps of VZV is gE, followed by gB, gH and gI. Glycoprotein gE (encoded by ORF68) is essential for VZV replication and cell-to-cell spread in the skin, and glycoproteins gB (encoded by ORF31) and gH (encoded by ORF37) regulate virus entry into cells and cell-to-cell fusion to produce syncytia. In addition, gI and gL are molecular chaperones of gE and gH, respectively, each with the function of activating gE and gH, and jointly participate in the above-mentioned cell fusion and syncytia production. The production of syncytia is an important sign of VZV infection. Syncytia at early time points (24 and 48 hpi) are first labeled with gE antibodies; by 48 hpi, both VZV gE and gH can be detected. Unlike gE, gC has delayed expression in VZV infection and can only be detected at 72 and 96 hpi. Therefore, the detection of highly immunogenic, highly conserved and characteristic glycoproteins is of great value in the diagnosis of VZV infection.

[0004] There are many Elisa detection methods based on VZV antibodies, such as the double antibody sandwich method, but all of them have problems such as low sample antibody titer, high detection threshold, complex experimental process, high time cost (>5 hours) and poor batch result uniformity, which limit their widespread clinical use. The indirect ELISA method is to pre-coat the known antigen, add the sample to form an antigen-antibody complex, add enzyme-labeled secondary antibody for signal amplification, and then detect the target antibody in the sample. Due to its high sensitivity, economy and flexibility, it has gradually been widely used in medical biological detection and scientific research. In the indirect ELISA method, the purification technology of antigen protein is the key to determining the sensitivity and specificity of detection. Currently, the expression and purification systems commonly used for viral glycoproteins include: mammalian cell expression system (such as CHO, HEK293 cells), yeast expression system (such as Pichia pastoris), prokaryotic expression system (such as Escherichia coli) and chromatography. Among them, the cell expression method has a high cost. Although Pichia pastoris and Escherichia coli have low costs and simple operations, they cannot correctly fold and glycosylate proteins, resulting in low activity. Insect cell expression systems (such as Sf9 / Sf21 cells) are particularly suitable for glycoproteins that require complex glycosylation modifications, such as herpes zoster virus glycoproteins, because they can perform more complex post-translational modifications.

[0005] VZV infection will induce the body to produce a strong antibody response, including IgM, IgA and IgG, especially against the glycoproteins on the virus shell. gE, gB, gI, gH, etc. are the main glycoproteins of VZV, which can trigger the production of IgM, IgA and IgG antibodies. Among them, IgM antibodies usually appear in the early stage of viral infection, indicating active VZV infection, and are the main antibody type for primary immune response, VZV reinfection or reactivation; IgG antibodies persist for a long time after infection, and may even be carried for life, and are used to guide and evaluate vaccination. Existing ELISA kits can usually only detect IgM or IgG antibodies alone, which to a certain extent limits its comprehensiveness and accuracy in the diagnosis of varicella-zoster virus infection, and cannot accurately determine the stage of infection. Summary of the invention

[0006] The purpose of the present invention is to provide a varicella-zoster virus combined diagnostic test kit to solve the above-mentioned deficiencies in the prior art.

[0007] In order to achieve the above-mentioned purpose, the present invention provides the following technical scheme: a varicella-zoster virus combined diagnostic detection kit, comprising a 96-well plate, an HRP enzyme-labeled secondary antibody, a substrate solution, a stop solution, a washing solution, a sample diluent, a positive control, and a negative control.

[0008] Furthermore, the 96-well plate is pre-packed with recombinant herpes zoster virus glycoprotein, and the recombinant herpes zoster virus glycoprotein is expressed and purified by an insect cell expression system.

[0009] Furthermore, the recombinant herpes zoster virus glycoprotein is at least three of gE, gB, gH, gC, gI, and gL.

[0010] Furthermore, the sample diluent is PBST containing 4% skim milk powder; the HRP enzyme-labeled secondary antibody is anti-human IgG monoclonal antibody and anti-human IgM monoclonal antibody; the washing solution is PBS containing 0.01 mol / L Tween-20 at a volume concentration of 0.05% and a pH of 7.2-7.4.

[0011] Furthermore, the substrate solution is a diaminobenzidine solution.

[0012] Furthermore, the stop solution is 1 mol / L H2SO4 solution.

[0013] Furthermore, the positive control solution is prepared by adding positive serum to the sample diluent at a ratio of 1:30 to 1:50, and the absorbance value of the positive control solution is greater than 1.0.

[0014] Furthermore, the negative control serum is prepared by adding negative serum to the sample diluent at a ratio of 5 wt %, and the absorbance value of the negative control solution is less than 0.1.

[0015] Compared with the prior art, the varicella-zoster virus combined diagnostic test kit provided by the present invention combines IgM and IgG for detection, which can more comprehensively reflect the infection status of varicella-zoster virus, and thus is closer to clinical needs. In the early stage of viral infection, a positive result of IgM indicates that the patient may be in an acute or active infection stage, which is of great significance for timely diagnosis and treatment. As the infection progresses, the gradual increase of IgG indicates that the body has produced a long-term immune response, which is crucial for evaluating the patient's immune status, predicting the development trend of the disease, and formulating subsequent treatment plans.

[0016] The detection method uses VZV capsid glycoprotein as the detection target, and uses VZV glycoproteins gE, gB, gH, gC, gI and gL expressed by an insect cell expression system, with a purity of more than 90%. Compared with the purification of specific antigens from VZV infected cells (cell expression system), it is easier to obtain a large amount of high-purity recombinant VZV glycoproteins through the insect cell expression system. In addition, using at least three of the recombinant high-purity VZV gE, gB, gH, gC, gI and gL glycoproteins as coating antigens can significantly reduce the differences between coating plates.

[0017] In addition, the combined detection of IgM and IgG can also be used to distinguish between primary infection and recurrent infection. In primary infection, the antibody levels of IgM and IgG usually increase at the same time, but the increase of IgM is often more rapid and significant; in recurrent infection, because the body already has immune memory against the virus, the antibody level of IgG may increase rapidly, while the increase of IgM is relatively insignificant.

[0018] Therefore, the combined detection of IgM and IgG against VZV viral glycoprotein not only improves the accuracy of varicella-zoster virus infection detection, but also provides more comprehensive and reliable information for clinical diagnosis and treatment, which is of great significance for guiding clinical medication, evaluating treatment effects and preventing disease transmission. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0020] Figure 1 A flow chart showing the principle of the kit provided by an embodiment of the present invention;

[0021] Figure 2 A schematic flow chart of a method for using the kit provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0022] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0023] Embodiment 1:

[0024] See also Figure 1 A varicella-zoster virus combined diagnostic test kit includes a 96-well plate, an HRP enzyme-labeled secondary antibody, a substrate solution, a stop solution, a washing solution, a sample diluent, a positive control, and a negative control.

[0025] The 96-well plate is pre-packed with recombinant herpes zoster virus glycoprotein, which is expressed and purified by an insect cell expression system.

[0026] The recombinant herpes zoster virus glycoproteins are at least three of gE, gB, gH, gC, gI, and gL.

[0027] The sample diluent is PBST containing 4% skim milk powder; the HRP enzyme-labeled secondary antibodies are anti-human IgG monoclonal antibodies and anti-human IgM monoclonal antibodies; the washing solution is PBS containing 0.01 mol / L Tween-20 with a volume concentration of 0.05% and a pH of 7.2-7.4.

[0028] The enzyme substrate solution is diaminobenzidine solution.

[0029] The stop solution is 1 mol / L H2SO4 solution.

[0030] The positive control solution is prepared by adding positive serum to the sample diluent at a ratio of 1:30 to 1:50, and the absorbance value of the positive control solution is greater than 1.0; the negative control serum is prepared by adding negative serum to the sample diluent at a ratio of 5wt%, and the absorbance value of the negative control solution is less than 0.1.

[0031] The specific implementation method is that the 96-well plate is pre-coated with recombinant herpes zoster virus glycoprotein: the 96-well plate is the basis of the detection, and at least three of the recombinant herpes zoster virus glycoproteins gE, gB, gH, gC, gI, and gL are pre-coated on it. When the antibodies in the sample to be tested bind to these antigens, antigen-antibody complexes can be formed, providing a basis for subsequent detection steps.

[0032] Using VZV capsid glycoprotein as the detection antigen, the purity of VZV glycoprotein expressed by the insect cell expression system can reach more than 90%. Compared with purifying specific antigens from VZV infected cells, it is easier to obtain a large amount of high-purity recombinant VZV glycoprotein through the insect cell expression system. In addition, using recombinant high-purity VZV glycoprotein as the coating antigen can significantly reduce the difference between coated plates.

[0033] The recombinant herpes zoster virus glycoprotein was expressed and purified by an insect cell expression system, and the gene sequences of VZV glycoprotein gE, gB, gH, gC, gI and gL were cloned into the rod-shaped expression vector pAcgp67B respectively. The recombinant vector and the linear baculovirus were co-transfected into the insect cell Sf9 cultured in serum-free medium by homologous recombination. After 12 hours, the medium was replaced with a serum-containing medium for continued culture, and the recombinant vector and the linear baculovirus underwent homologous recombination, thereby obtaining the recombinant baculovirus 1;

[0034] The recombinant baculovirus 1 was added to a 10 cm cell plate filled with cells and containing serum culture medium to amplify the virus, and the recombinant baculovirus 2 was harvested; the recombinant baculovirus 2 was added to a 100 ml conical flask containing cells to expand the virus culture, and the recombinant baculovirus 3 was harvested after 3 to 4 days;

[0035] High Five TM insect cells were infected with recombinant baculovirus 3. After 4-5 days, the infected insect cells and their culture supernatant were harvested. The harvested cell supernatant was stained with a retentate molecule with a relative molecular mass of 30×10 3 The supernatant was concentrated by tangential flow ultrafiltration membrane cells, and then dialyzed with PBS. After 12 hours, the insoluble matter was removed by centrifugation. The target protein was purified using Co medium and affinity chromatography, and the target protein was eluted with 150 mmol / L imidazole. The purified VZV glycoprotein was stored in PBS.

[0036] HRP enzyme-labeled secondary antibodies (anti-human IgG monoclonal antibodies and anti-human IgM monoclonal antibodies): These antibodies are used to detect specific antibodies (IgG and IgM) in the sample to be tested. They can bind to IgG and IgM in the sample to be tested, amplify the signal, and make it easier to detect; HRP (horseradish peroxidase) is an enzyme marker that can catalyze the color development reaction of the substrate solution through the enzyme activity of HRP, thereby realizing the detection of antibodies.

[0037] Substrate solution (diaminobenzidine solution): The substrate solution undergoes a color reaction under the catalysis of HRP. Diaminobenzidine is a commonly used substrate that can be oxidized and colored under the action of HRP, thereby providing intuitive detection results.

[0038] Stop solution (1mol / L H2SO4 solution): The stop solution is used to stop the color development reaction of the substrate solution. When the color development reaction reaches a certain level, adding the stop solution can stop the reaction, thereby fixing the color development result for easy observation and recording.

[0039] Washing solution (PBS with a volume concentration of 0.05% Tween-20, 0.01 mol / L and pH 7.2-7.4): Washing solution is used to wash the ELISA plate during the detection process to remove unbound antibodies and other impurities. Tween-20 is a surfactant that can increase the washing effect, while PBS provides a suitable pH value and ionic strength environment.

[0040] Sample diluent: The sample diluent is used to dilute the sample to be tested to a moderate concentration for easy detection; the sample diluent is PBST containing 4% skimmed milk powder by mass. The sample diluent is prepared as follows: weigh 4 g of skimmed milk powder, add the weighed skimmed milk powder to 80 ml of PBST solution, use a magnetic stirrer to help dissolve the skimmed milk powder, ensure that the skimmed milk powder has no lumps and is completely dissolved, then use PBST solution to add to 100 ml, mix well to obtain the sample diluent.

[0041] Positive control and negative control: The positive control solution and negative control solution are used to verify the effectiveness and accuracy of the detection system, respectively. The positive control solution should contain known varicella-zoster virus antibodies and produce an absorbance value greater than 1.0; the negative control solution should not contain varicella-zoster virus antibodies and produce an absorbance value less than 0.1. By comparing the absorbance value of the sample to be tested with the absorbance values ​​of the positive control and negative control, it can be determined whether the sample to be tested contains varicella-zoster virus antibodies.

[0042] Embodiment 2:

[0043] See also Figure 2 This embodiment provides a technical solution based on the first embodiment: a method for using a varicella-zoster virus combined diagnostic test kit.

[0044] S1. Before use, gently invert all reagents in the kit to mix thoroughly.

[0045] Thorough mixing of reagents is the first step to ensure the success of the experiment. Reagents in ELISA kits, such as antibodies, substrates, washing solutions, etc., may be separated or precipitated during storage due to gravity, temperature fluctuations or long-term standing.

[0046] Therefore, all reagents must be fully mixed before use to ensure uniform concentrations of each component, thereby avoiding experimental errors; mixing reagents can ensure that the concentrations of each component remain consistent each time the sample is added, improving the accuracy and repeatability of the experiment; the degree of mixing of reagents directly affects the reliability of the experimental results. If the reagents are not fully mixed, the test results of some wells may be too high or too low, thus affecting the accuracy of the entire experiment.

[0047] S2. Determine the number of strips required based on the number of samples to be tested plus the number of standards, and make duplicate wells for each standard and blank well.

[0048] At least two wells are made for each sample to be tested for subsequent experimental detection; the standard is used to establish a concentration-OD value curve to calculate the concentration of the sample to be tested; the blank well is used to detect background signals, that is, signals generated by nonspecific binding or instrument errors; setting up duplicate wells for each standard and blank well can further improve the accuracy and reliability of the experiment.

[0049] By setting standards and blank wells, you can calibrate the experimental conditions, reduce experimental errors, and improve the accuracy and reliability of experimental results. Standards and blank wells are an indispensable part of ELISA experiments. They provide an important reference for the interpretation of experimental results and help to accurately determine the concentration of the sample to be tested.

[0050] S3. Dilute the sample to be tested with sample diluent at a ratio of 1:1 (can be adjusted according to experimental conditions) and add 50ul into the reaction well.

[0051] Sample diluent is used to optimize reaction conditions and improve detection sensitivity and accuracy; adding the diluted specimen to the reaction well can ensure that the binding reaction between the antigen and the antibody is carried out within an appropriate concentration range.

[0052] The sample diluent can reduce the interference of high-concentration antigen on the antibody binding reaction, improve the sensitivity and accuracy of the detection, and at the same time, the diluted sample is easier to bind to the antibody to form a stable antigen-antibody complex.

[0053] Sample dilution is a key step in ELISA experiments. If the sample concentration is too high, it may lead to antibody binding saturation, thereby reducing the sensitivity of the test; while appropriate dilution can ensure the full binding of antigen and antibody and improve the accuracy of the experimental results.

[0054] S4. Add 50ul of the diluted standard to the empty reaction wells, add 50ul of each sample to be tested to the other two empty reaction wells, and then add 50ul of anti-human IgG monoclonal antibody to one well and 50ul of anti-human IgM monoclonal antibody to the other well. Cover the membrane plate, shake gently to mix, and incubate at 37℃ for 1 hour.

[0055] HRP enzyme-labeled secondary antibody (anti-human IgG monoclonal antibody and anti-human IgM monoclonal antibody), HRP as an enzyme label can catalyze the substrate to produce a color reaction, providing a basis for subsequent color reactions.

[0056] S5. After incubation, shake off the liquid in the wells, fill each well with washing solution, shake for 30 seconds, shake off the washing solution, pat dry with absorbent paper, and repeat this operation 3 times.

[0057] The purpose of washing is to remove impurities and excess antibodies that are not bound to the streptavidin-HRP to reduce background interference and further improve the specificity and sensitivity of the test. If the washing is insufficient or the number of washings is insufficient, unbound components may remain, thus affecting the accuracy of the experimental results.

[0058] S6. After washing, add 50ul of substrate diaminobenzidine solution to each well, shake gently to mix, and incubate at 37°C for 10 minutes, avoiding light.

[0059] Diaminobenzidine (DAB) is a commonly used ELISA substrate, which can undergo a color reaction under the catalysis of HRP to generate a colored product. The colored product has a maximum absorption peak at a specific wavelength and can be used to determine the OD value.

[0060] Adding substrate can trigger a color reaction, and measuring the OD value can reflect the amount of antigen. Incubation helps to accelerate the color reaction and make the color more stable.

[0061] The color reaction is one of the key steps to achieve quantitative detection in ELISA experiments. If the color reaction is insufficient or the color is unstable, it will directly affect the determination of the OD value and the accuracy of the experimental results. Therefore, the incubation step after adding the substrate and avoiding light exposure are both crucial.

[0062] S7. After incubation, remove the ELISA plate and quickly add 50ul of stop solution.

[0063] The stop solution can stop the color development reaction and stabilize the color. After the stop solution is added, the color development reaction stops immediately. The color depth at this time is proportional to the amount of HRP, thus reflecting the amount of antigen. Adding the stop solution can ensure that the color development reaction stops within an appropriate time, stabilizes the color, and facilitates the subsequent determination of the OD value.

[0064] The timeliness and accuracy of terminating the reaction are crucial to the accuracy of the experimental results. If the reaction is not terminated in time or incompletely, the color may continue to change, thus affecting the determination of the OD value and the accuracy of the experimental results.

[0065] S8. After adding the stop solution, immediately measure the OD value of each well at a wavelength of 450 nm and record the OD value data.

[0066] The colored product has a maximum absorption peak at a wavelength of 450nm. The OD value at this wavelength can reflect the amount of the colored product, thereby reflecting the amount of the antigen. The OD value is proportional to the concentration of the colored product, so it can be used for quantitative detection.

[0067] Determining the OD value is the last step in achieving quantitative detection in the ELISA experiment. By comparing the OD value of the sample to be tested with the OD value of the standard, the concentration of the sample to be tested can be calculated. Determining the OD value is one of the most critical steps in the ELISA experiment. If the determination is inaccurate or there is an error, it will directly affect the accuracy and reliability of the experimental results. Therefore, when determining the OD value, the accuracy and stability of the instrument must be ensured, and the correct operating procedures and precautions must be followed.

[0068] Each step of the ELISA kit operation is crucial. From the preparation and addition of reagents to the addition and incubation of antibodies, to washing, the addition of substrates and color development, as well as the termination of reactions and the determination of OD values, each step is closely linked and together constitutes the core process of the ELISA experiment. In this process, the reaction principle, function and importance of each step are fully reflected. Only by strictly following the operation steps can the accuracy and reliability of the experimental results be ensured. Therefore, when conducting an ELISA experiment, strictly follow the operation steps and pay attention to observe the changes during the experiment so as to discover and solve problems in a timely manner; at the same time, the experimenter should also have good experimental skills and a rigorous experimental attitude to ensure the accuracy and reliability of the experimental results.

[0069] The above description is only by way of illustration of certain exemplary embodiments of the present invention. It is undoubted that those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A varicella-zoster virus combined diagnostic test kit, characterized in that: Includes 96-well plate, HRP enzyme-labeled secondary antibody, substrate solution, stop solution, washing solution, sample diluent, positive control, and negative control.

2. A varicella-zoster virus combined diagnostic test kit according to claim 1, characterized in that: The 96-well plate is pre-packed with recombinant herpes zoster virus glycoprotein, and the recombinant herpes zoster virus glycoprotein is expressed and purified by an insect cell expression system.

3. A varicella-zoster virus combined diagnostic test kit according to claim 2, characterized in that: The recombinant herpes zoster virus glycoprotein is at least three of gE, gB, gH, gC, gI, and gL.

4. A varicella-zoster virus combined diagnostic test kit according to claim 1, characterized in that: The sample diluent is PBST containing 4% skim milk powder; the HRP enzyme-labeled secondary antibodies are anti-human IgG monoclonal antibody and anti-human IgM monoclonal antibody; the washing solution is PBS containing 0.01 mol / L of 0.05% Tween-20 and pH 7.2-7.

4.

5. A varicella-zoster virus combined diagnostic test kit according to claim 1, characterized in that: The substrate solution is a diaminobenzidine solution.

6. A varicella-zoster virus combined diagnostic test kit according to claim 1, characterized in that: The stop solution is 1 mol / L H2SO4 solution.

7. A varicella-zoster virus combined diagnostic test kit according to claim 1, characterized in that: The positive control solution is prepared by adding positive serum to the sample diluent at a ratio of 1:30 to 1:50, and the absorbance value of the positive control solution is greater than 1.

0.

8. A varicella-zoster virus combined diagnostic test kit according to claim 1, characterized in that: The negative control serum is prepared by adding negative serum in a proportion of 5 wt % into the sample diluent, and the absorbance value of the negative control solution is less than 0.1.

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