Reagents for detecting varicella-zoster virus infection and methods of making same

Abstract

The present application relates to the field of immunological detection, and particularly relates to a reagent for detecting varicella-zoster virus infection and a preparation method thereof. The reagent comprises R1 reagent and R2 reagent; the R1 reagent is a working solution of streptavidin magnetic beads, and the preparation method comprises the following steps: firstly coupling activated carboxyl magnetic beads with streptavidin; dialyzing the magnetic beads obtained by the first coupling to remove streptavidin physically combined with the magnetic beads; secondly coupling the dialyzed magnetic beads with streptavidin; quenching the magnetic beads obtained by the second coupling, and then placing the magnetic beads in a working solution to obtain the working solution; the working solution contains fetal bovine serum, bovine serum albumin and lysine; the R2 reagent is a biotinylated VZV gE recombinant protein, and the VZV gE recombinant protein is a varicella-zoster virus gE glycoprotein extracellular domain fragment expressed by human embryonic kidney cells. The reagent has high sensitivity and specificity in the detection of varicella-zoster virus infection.

Description

Technical Field

[0001] This invention relates to the field of immunoassay detection, specifically to a reagent for detecting varicella-zoster virus infection and its preparation method. Background Technology

[0002] Varicella-zoster virus (VZV), herpes simplex virus (HSV), cytomegalovirus (CMV), and Epstein-Barr virus (EBV) all belong to the herpesvirus genus and are pathogenic to humans. VZV is a double-stranded DNA virus belonging to the alphaherpesvirus subfamily. Humans are its only natural host and it exhibits neurotropism. VZV is primarily transmitted through droplets (contaminated aerosols) or contaminated objects (such as virus-containing vesicles or eschars). It is the common pathogen for both clinical varicella and shingles. The VZV genome contains 71 genes encoding 67 different proteins, including six glycoproteins (gpI–gpVI), now uniformly named gE, gB, gH, gI, gC, and gL. Glycoproteins gE, gB, and gH are extremely abundant in infected cells and are also present in the virion's cell membrane. The gE protein consists of 623 amino acids and is encoded by the ORF68 gene, which is a 1872-base gene located in a short segment of the VZV genome. Its N-terminus is a 544-amino acid hydrophilic extracellular region (containing a signal peptide), and its C-terminus consists of a 17-amino acid transmembrane hydrophobic region and a 62-amino acid intracellular region. gE is a type I membrane protein and an essential glycoprotein for the generation of infectious VZV particles. It is also the most immunogenic and abundant glycoprotein on the viral envelope and infected cell membrane.

[0003] There are two types of VZV infection in humans: primary infection with chickenpox (varicella) and recurrent infection with shingles (zoster). When a patient is first infected with VZV, they will experience chickenpox symptoms. After recovery, the virus remains dormant in the nerve cells of the sensory ganglia for life. Years later, when the host is elderly or their immunity declines, the dormant virus may reactivate and proliferate, damaging skin cells and local nerves, forming shingles. If chickenpox is not diagnosed and treated promptly, it can lead to secondary complications such as varicella pneumonia, encephalitis, and myocarditis, and even death. Shingles causes prolonged and severe pain, significantly impacting patients' lives and burdening public health expenditures. In recent years, the incidence of chickenpox and shingles has been on the rise, with reports of severe chickenpox resulting in death worldwide. Furthermore, it is prone to causing outbreaks in densely populated areas.

[0004] VZV is one of the most common viral infections, and blood tests are helpful for early diagnosis. Currently, clinical diagnosis of VZV infection mainly relies on typical clinical symptoms and skin lesions. However, other viruses (such as Coxsackievirus and Herpes Simplex Virus) can also cause varicella-like rashes. Therefore, relying solely on typical clinical symptoms and skin lesions to diagnose VZV infection can easily lead to missed or misdiagnosis. Laboratory tests are necessary for auxiliary diagnosis, with definitive confirmation depending on virus isolation from herpes fluid or a positive serum-specific IgM antibody test. Since virus isolation from herpes fluid is too complex and time-consuming, the most commonly used method in clinical practice is the serum-specific IgM antibody detection method.

[0005] Magnetic particle chemiluminescence immunoassay is an emerging analytical method that combines magnetic separation, chemiluminescence, and immunoassay techniques. This technology fully leverages the speed and ease of automation of magnetic separation, the high sensitivity of chemiluminescence, and the specificity of immunoassay, demonstrating exceptional performance in the field of bioanalysis. Magnetic particle chemiluminescence is a cutting-edge technology in immunoassay reagents, but it has high technical barriers; its core technology lies in the coupling of magnetic beads with antigens.

[0006] Streptavidin is a protein secreted by Streptomyces that specifically binds to biotin, exhibiting the strongest known non-covalent binding force. Due to this property, the streptavidin-biotin reaction system has been widely used in purification and detection. Streptavidin exists as a homotetramer, with each mole of tetramer molecule binding four moles of biotin. Therefore, it has a signal amplification effect in immunoassay, further improving the sensitivity of the immunoassay. However, the coupling of streptavidin with magnetic beads often suffers from low coupling efficiency due to physical adsorption, thus affecting the final sensitivity of the magnetic beads.

[0007] Furthermore, research has found that interference in immunoassay techniques can affect the specificity, accuracy, and sensitivity of reagent detection. The main causes of immunoassay interference include interference from reagent components (e.g., solid-phase carriers, chemiluminescent substances), interference from the components of the sample to be tested, and interference caused by the detection method itself. In magnetic microparticle chemiluminescent immunoassay, interference from reagent components mainly stems from non-specific binding between the streptavidin-coated solid-phase carrier and certain proteins or hydrophobic analytes in the sample to be tested. After streptavidin-labeled magnetic beads, inert proteins are typically used to block them to prevent non-specific adsorption from vacant epitopes. However, due to the steric hindrance of streptavidin on the surface of the beads, the inert proteins cannot effectively block the streptavidin beads. Therefore, during use, the streptavidin beads can non-specifically bind to biotinylate antigens, acrid ester antibodies, etc., leading to increased non-specific background and affecting detection results and product performance.

[0008] Therefore, new diagnostic reagents need to be developed to improve the accuracy and sensitivity of VZV infection diagnosis. Summary of the Invention

[0009] To overcome the shortcomings in the above-mentioned fields, the present invention provides a reagent for detecting varicella-zoster virus infection, which has high sensitivity and high specificity.

[0010] The reagents provided by this invention for detecting varicella-zoster virus infection include reagent R1 and reagent R2.

[0011] The R1 reagent is a streptavidin magnetic bead working solution, which is prepared by: first coupling activated carboxyl magnetic beads with streptavidin; dialyzing the magnetic beads obtained from the first coupling to remove streptavidin physically bound to the magnetic beads; second coupling the dialyzed magnetic beads with streptavidin; quenching the magnetic beads obtained from the second coupling, and then placing them in the working solution to obtain the solution; the working solution contains fetal bovine serum, bovine serum albumin, and lysine.

[0012] The R2 reagent is a biotinylated VZV gE recombinant protein, which is an extracellular domain fragment of the varicella-zoster virus gE glycoprotein expressed in human embryonic kidney cells.

[0013] In some embodiments of the present invention, the carboxyl magnetic beads are nano magnetic beads with a particle size of 1 μm.

[0014] In some embodiments of the present invention, 0.01-0.03M phosphate buffer with pH 7.2-7.6 is used as the dialysate to dialyze the magnetic beads obtained in the first coupling.

[0015] In some embodiments of the present invention, the streptavidin magnetic beads obtained from the first coupling are placed in a microdialysis device for dialysis, and the dialysis solution is 0.01M phosphate buffer at pH 7.4. The dialysis solution is changed every 4 hours, and the device is magnetically stirred for 24 hours. After dialysis, the microdialysis device is centrifuged in a centrifuge tube to obtain the dialyzed streptavidin magnetic beads.

[0016] In some embodiments of the present invention, the dialyzed streptavidin magnetic beads are mixed with streptavidin coupling buffer, and after being gently stirred at 4°C for 12 hours, the magnetic beads are sorted and washed three times with 1 ml of 0.01 M phosphate buffer at pH 7.4 to obtain streptavidin magnetic beads after the second coupling.

[0017] In some embodiments of the present invention, the streptavidin magnetic beads obtained from the second coupling are quenched using a quenching buffer composed of 2-hydroxyethylamine and 0.01-0.03M phosphate buffer at pH 7.2-7.6.

[0018] In some embodiments of the present invention, quenching buffer is added to the streptavidin magnetic beads obtained from the second coupling, the mixture is gently mixed, and incubated on a vortex mixer for 45 min. The magnetic beads are collected using a magnetic rack, and the supernatant is removed. The magnetic beads are stored in 20 mM phosphate buffer at pH 7.4 to obtain a streptavidin magnetic bead suspension.

[0019] In some embodiments of the present invention, the human embryonic kidney cells are HEK293F cells.

[0020] In some embodiments of the present invention, the amino acid sequence of the extracellular domain fragment of the varicella-zoster virus gE glycoprotein is shown in SEQ ID NO: 1.

[0021] In some embodiments of the present invention, the working solution is an 18-22 mM phosphate buffer solution with a pH of 7.2-7.6 containing 0.2%-0.4% (v / v) fetal bovine serum, 4-6 g / ml bovine serum albumin and 0.1-0.3 g / ml lysine.

[0022] In some embodiments of the present invention, the reagent for detecting varicella-zoster virus infection further includes R3 reagent; the R3 reagent is an acridinium ester-labeled anti-human IgM antibody.

[0023] The present invention also provides a method for preparing any of the reagents described above for detecting varicella-zoster virus infection, comprising:

[0024] S1: The activated carboxyl magnetic beads are first coupled with streptavidin; the magnetic beads obtained from the first coupling are dialyzed to remove the streptavidin physically bound to the magnetic beads; the dialyzed magnetic beads are second coupled with streptavidin; the magnetic beads obtained from the second coupling are quenched and then placed in a working solution to obtain a streptavidin magnetic bead working solution; the working solution contains fetal bovine serum, bovine serum albumin and lysine;

[0025] S2: The extracellular domain fragment of varicella-zoster virus gE glycoprotein was expressed using human embryonic kidney cells, and the fragment was labeled with biotin to obtain biotinylated VZV gE recombinant protein.

[0026] In the above method, 0.01-0.03M phosphate buffer with pH 7.2-7.6 can be used as the dialysis solution to dialyze the magnetic beads obtained in the first coupling.

[0027] In the above method, the magnetic beads obtained from the second coupling can be quenched using a quenching buffer, which is composed of 2-hydroxyethylamine and 0.01-0.03M phosphate buffer at pH 7.2-7.6.

[0028] In the above method, the human embryonic kidney cells can be HEK293F cells.

[0029] In the above method, the amino acid sequence of the extracellular domain fragment of the varicella-zoster virus gE glycoprotein is shown in SEQ ID NO: 1.

[0030] In the above method, the working solution is preferably an 18-22mM phosphate buffer solution with a pH of 7.2-7.6 containing 0.2%-0.4% (v / v) fetal bovine serum, 4-6g / ml bovine serum albumin and 0.1-0.3g / ml lysine.

[0031] In some embodiments of the present invention, the working solution is a 20 mM phosphate buffer at pH 7.4 containing 0.3% (v / v) fetal bovine serum, 5 g / ml bovine serum albumin, 0.1 g / ml lysine and 0.1% (v / v) Proclin-300 preservative.

[0032] In some embodiments of the present invention, the steps for preparing the extracellular domain fragment of the varicella-zoster virus gE glycoprotein are as follows:

[0033] (1) Using varicella-zoster virus genomic DNA as a template, the coding gene of the extracellular domain fragment of varicella-zoster virus gE glycoprotein was amplified by PCR.

[0034] (2) The amplified coding gene was inserted into a eukaryotic expression vector to obtain a recombinant vector carrying the coding gene for the extracellular domain fragment of the varicella-zoster virus gE glycoprotein.

[0035] (3) The constructed recombinant vector was transfected into human embryonic kidney cells to obtain recombinant cells;

[0036] (4) Culture the recombinant cells and extract and purify the extracellular domain fragment of the varicella-zoster virus gE glycoprotein.

[0037] In some embodiments of the present invention, the nucleotide sequence of the gene encoding the extracellular domain fragment of the varicella-zoster virus gE glycoprotein is shown in SEQ ID NO: 2.

[0038] In some embodiments of the present invention, the eukaryotic expression vector used is pcDNA3.1 / myc-His(-)B plasmid.

[0039] The present invention also provides a kit comprising any of the reagents described herein for detecting varicella-zoster virus infection.

[0040] In the kit, the preferred concentration of streptavidin magnetic beads in reagent R1 is 0.5 mg / ml.

[0041] In some embodiments, a method for detecting varicella-zoster virus infection using the reagents of the present invention includes: taking 20 μl of 0.5 mg / ml streptavidin magnetic bead working solution, 100 μl of 1.0 μg / ml biotinylated VZV gE recombinant protein, and 10 μl of blood sample (serum or plasma), mixing them, and reacting at 37°C for 10 min; after washing three times, adding 100 μl of 0.05 mg / ml acridinium ester-labeled mouse anti-human IgM antibody solution, and reacting at 37°C for 10 min; after washing three times, adding 100 μl of activation solution A and 100 μl of activation solution B, and measuring the luminescence value (RLU). Activation solution A is an aqueous solution containing 0.1 M HNO3 and 0.2% H2O2, and activation solution B is an aqueous solution containing 0.25 mol / L NaOH and 0.1% CTAC.

[0042] Using the reagents of this invention, varicella-zoster virus (VZV) gE glycoprotein IgM antibodies in human serum or plasma can be detected in vitro via an indirect method. VZV gE glycoprotein IgM antibodies are present in the serum or plasma of subjects infected with VZV. After mixing streptavidin magnetic bead working solution, biotinylated VZV gE recombinant protein, and a blood sample, the streptavidin magnetic beads specifically bind to the biotinylated VZV gE recombinant protein through the streptavidin-biotin interaction. The VZV gE glycoprotein IgM antibodies in the serum or plasma also specifically bind to the VZV gE recombinant protein, thereby forming a magnetic bead-IgM antibody complex. Anti-human IgM antibodies labeled with signaling molecules (e.g., acridinium ester) are bound to magnetic beads-IgM antibody complexes to form magnetic bead-IgM antibody-signaling molecule complexes. After adding the appropriate signal exciter, the luminescence value (RLU) is measured. The luminescence value can be used to determine the content of VZV gE glycoprotein IgM antibody in human serum or plasma, thereby assisting in the diagnosis of VZV infection status.

[0043] The reagent for detecting varicella-zoster virus infection provided by this invention has the following advantages:

[0044] High sensitivityAfter the initial binding of streptavidin to the magnetic beads, the physically adsorbed streptavidin is removed by dialysis. Then, the dialyzed streptavidin magnetic beads are bound to streptavidin a second time, ensuring that sufficient streptavidin is stably bound to the surface of the magnetic beads. This also prevents the streptavidin from being washed away during the subsequent cleaning steps of the fully automated chemiluminescence analyzer, resulting in an increased magnetic bead signal value and extremely high sensitivity.

[0045] High specificity ① Since humans are the only host for varicella-zoster virus, the extracellular domain of the varicella-zoster virus gE glycoprotein expressed by human embryonic kidney cells most closely resembles the natural conformation of the viral protein and can specifically bind to gE glycoprotein antibodies in blood samples. ② A specialized working solution was designed for the streptavidin-magnetic bead coupling method of this invention. This working solution effectively blocks vacant epitopes on the surface of the magnetic beads, reducing the exposure of numerous small epitopes on the magnetic bead surface caused by the double-linking of streptavidin, avoiding high background levels due to non-specific adsorption, reducing background values, and improving the specificity of magnetic bead detection. Attached Figure Description

[0046] Figure 1 The results show the PCR amplification of the gene encoding the extracellular domain fragment of VZV gE glycoprotein; lane M is the DNA marker, lane 1 is the amplified gene encoding the extracellular domain fragment of VZV gE glycoprotein, and lane 2 is the negative control (the product of PCR performed using ddH2O as a template).

[0047] Figure 2 The results of double enzyme digestion identification of recombinant plasmid gE-pcDNA3.1 / myc-His(-) are shown; lane M is the DNA marker, and lane 1 is the Hind III and Xhol I double enzyme digestion product of recombinant plasmid gE-pcDNA3.1 / myc-His(-).

[0048] Figure 3 The results of RT-PCR detection of mRNA expression of the gene encoding the extracellular domain fragment of VZV gE glycoprotein are shown. Lane M is the DNA Marker, lane 1 is the RT-PCR product of HEK293F cells transfected with the empty vector pcDNA3.1 / myc-His(-)B, lane 2 is the RT-PCR product of HEK293F cells transfected with gE-pcDNA3.1 / myc-His(-), and lane 3 is the RT-PCR product of normal HEK293F cells.

[0049] Figure 4The results of SDS-PAGE identification of purified VZV gE recombinant protein are shown; lane M is the protein marker, lane 1 is the purified VZV gE recombinant protein, and lane 2 is the protein purification product of HEK293F cells transfected with pcDNA3.1 / myc-His(-)B empty vector. Detailed Implementation

[0050] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for explanation and illustration only and do not limit the scope of the present invention in any way.

[0051] Experimental materials used in the following examples:

[0052] Varicella-zoster virus (VZV): Extracted from the fluid of skin blisters in patients diagnosed with shingles at the Dermatology Clinic of Peking University International Hospital.

[0053] The pcDNA3.1 / myc-His(-)B vector is a mammalian expression vector purchased from Ubisoft Biotechnology Co., Ltd. This vector regulates exogenous gene expression via a CMV promoter, and carries both a myc tag and a His tag at the C-terminus. The vector is protected against ampicillin (Amp), and the selection marker is neomycin (Neo).

[0054] DH5a Escherichia coli competent cells were prepared in our laboratory and are commercially available.

[0055] HEK293F cells were purchased from Shanghai Mingjin Biotechnology Co., Ltd.

[0056] Carboxyl magnetic beads, with a particle size of 1 μm, were provided by Professor Hou Yanglong's research group at Peking University. These carboxyl magnetic beads are commercially available.

[0057] Streptavidin was purchased from SIGMA, Inc., USA.

[0058] DNA polymerase, dNTPs, and 10×PCR buffer were purchased from Tiangen Biotech (Beijing) Co., Ltd. T4 DNALigase and 10×ligation Buffer were purchased from Takara Bio Engineering (Dalian) Co., Ltd. NHS, EDC, EDC.HCl, and MES were purchased from Sinopharm Group.

[0059] 0.01M phosphate buffer (pH 7.4): Weigh 8g NaCl, 0.2g KCl, 1.44g Na2HPO4 and 0.24g KH2PO4, dissolve them in 800ml distilled water, adjust the pH of the solution to 7.4 with HCl, and finally add distilled water to a final volume of 1L.

[0060] 20mM phosphate buffer (pH 7.4): Dilute 100mL of 0.2M PB (Solution A:Solution B = 31:69) with water to 1000mL. Solution A (0.2M sodium dihydrogen phosphate aqueous solution): Weigh 27.6g of NaH₂PO₄·H₂O, dissolve in ultrapure water, and bring the volume to 1L. Filter and store. Solution B (0.2M disodium hydrogen phosphate aqueous solution): Weigh 53.6g of Na₂HPO₄·7H₂O, dissolve in distilled water, and bring the volume to 1L.

[0061] Unless otherwise specified, the reagents used in the following examples are all conventional reagents in the art, commercially available or prepared according to conventional methods in the art, and are of laboratory purity. Unless otherwise specified, the experimental methods and conditions used in the following examples are conventional experimental methods and conditions in the art, and can be found in relevant experimental manuals, public literature, or manufacturer's instructions. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0062] Example 1. Preparation of varicella-zoster virus (VZV) antigen protein

[0063] 1. Amplification of the gene encoding the extracellular domain fragment of VZV gE glycoprotein

[0064] Specific primers gE-F and gE-R for encoding the extracellular domain fragment of the VZV gE glycoprotein (target gene) were designed and synthesized. Genomic DNA of varicella-zoster virus (VZV) was extracted using the TaKaRa MiniBEST Viral RNA / DNA Extraction Kit (catalog number 9766). Using the VZV genomic DNA as a template, the target gene was amplified by polymerase chain reaction (PCR) using primers gE-F and gE-R.

[0065] The nucleotide sequences of the primers are as follows:

[0066] gE-F:5'-AGGCAG ACCATGGGGACAGTTAATAAACCTGT-3' (Italicized text indicates HindIII restriction site);

[0067] gE-R:5'-AATAAT GGCATATCGTAGAAGTGGTGACG-3' (Italicized text indicates the Xhol I restriction site).

[0068] The nucleotide sequence (1617 bp) of the gene encoding the extracellular domain fragment of the VZV gE glycoprotein is as follows:

[0069]

[0070]

[0071] The amino acid sequence (539aa) of the extracellular domain fragment of the VZV gE glycoprotein is as follows:

[0072]

[0073] PCR reaction mixture (50 μl):

[0074]

[0075] The reaction conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 50 s; 60℃ annealing for 45 s; 72℃ extension for 2 min, for a total of 35 cycles; and a final extension at 72℃ for 10 min. After PCR amplification, 5 μl of the product was subjected to electrophoresis on a 1.0% agarose gel at 100 V for 35 min, stained with GelRed, and the results were photographed and analyzed.

[0076] The results are as follows Figure 1 As shown, lane 1 contains the amplified target gene fragment, with a specific band of 1617 bp visible; lane 2 is the negative control (the product of PCR performed using ddH2O as a template), with no band produced. Sequencing confirmed that the nucleotide sequence of the amplified target gene fragment is correct.

[0077] The target gene fragment was recovered using the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 according to the product instructions.

[0078] 2. Construction and identification of recombinant plasmid gE-pcDNA3.1 / myc-His(-)

[0079] The recovered target gene fragment and the pcDNA3.1 / myc-His(-)B vector were double-digested using Hind III and Xhol I restriction endonucleases (TaKaRa), respectively. The total digestion volume was 20 μl, including 16 μl template DNA, 1 μl Hind III and 1 μl Xhol I restriction endonucleases, and 2 μl 10× buffer. Digestion was carried out at 37℃ for 16 h.

[0080] The target gene fragment obtained by double enzyme digestion and the pcDNA3.1 / myc-His(-)B linear vector were recovered using the TaKaRa MiniBEST Agarose Gel DNA ExtTaction Kit Ver.4.0 gel recovery kit.

[0081] The digested target gene fragment was ligated with the pcDNA3.1 / myc-His(-)B linear vector using T4 DNA Ligase (TaKaRa). The total ligation volume was 10 μl, including 6 μl of the target gene fragment, 2 μl of the pcDNA3.1 / myc-His(-)B linear vector, 1 μl of 10× ligation buffer, and 1 μl of T4 DNA Ligase. Ligation was carried out overnight at 4°C.

[0082] The following day, the ligation product was used to transform DH5α competent *E. coli* cells, plated on LB agar plates containing 50 μg / ml Amp, and incubated upside down at 37°C for 16 h. Single colonies were picked, amplified, and plasmids were extracted. After identification by double digestion with Hind III and Xhol I, sequencing yielded the recombinant plasmid gE-pcDNA3.1 / myc-His(-) with the correct sequence. The Hind III and Xhol I double digestion results of the recombinant plasmid gE-pcDNA3.1 / myc-His(-) are shown below. Figure 2 As shown, the gene fragment and vector fragment are the correct sizes.

[0083] 3. Eukaryotic expression and identification of VZV gE fusion protein

[0084] HEK293F cells (Shanghai Mingjin) were transfected using Lipofectamine 2000 transfection reagent (Invitrogen) according to the instructions. The recombinant plasmid gE-pcDNA3.1 / myc-His(-) and the empty vector pcDNA3.1 / myc-His(-)B were transfected. Cells were cultured at 37°C using HEK293F complete culture medium (Shanghai Mingjin). Forty-eight hours after transfection, G418 antibiotic (Invitrogen) was added to the culture to a final concentration of 750 μg / ml for screening positive clones. Cells were cultured at 37°C for approximately 14 days, after which monoclonal cells were observed and expanded. Untransfected normal HEK293F cells served as a blank control.

[0085] (1) RT-PCR detection of mRNA expression of the target gene fragment

[0086] Total RNA was extracted from HEK293F cells transfected with the gE-pcDNA3.1 / myc-His(-) recombinant plasmid, HEK293F cells transfected with the pcDNA3.1 / myc-His(-)B empty vector (negative control), and normal HEK293F cells (blank control) using the Novizumi RNA-easy™ Isolation Reagent (Vazyme#R701) according to the product instructions. Then, using the HyperScript III 1st Strand cDNA Synthesis Kit (#R101) from Newbee Biotechnology, the extracted total RNA was reverse transcribed into cDNA using random primers according to the product instructions. PCR was then performed according to the following system and procedure to detect the expression of the target gene fragment's mRNA.

[0087] PCR reaction mixture (50 μl):

[0088]

[0089] The reaction conditions were: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 50 s; 55℃ annealing for 45 s; 72℃ extension for 2 min; for a total of 35 cycles; and a final extension at 72℃ for 10 min. After PCR amplification, 5 μl of the product was subjected to electrophoresis on a 1.0% agarose gel at 100 V for 35 min, stained with GelRed, and the results were photographed and analyzed.

[0090] The results are as follows Figure 3 As shown, a specific target band of 1617 bp was amplified from HEK293F cells stably transfected with the gE-pcDNA3.1 / myc-His(-) recombinant plasmid. No band was amplified from HEK293F cells transfected with the pcDNA3.1 / myc-His(-)B empty vector (negative control) or normal HEK293F cells (blank control). Monoclonal cells with high expression of the target gene were selected for amplification culture to obtain a cell suspension expressing the VZV gE recombinant protein.

[0091] (2) Purification and identification of VZV gE recombinant protein

[0092] Cell suspensions expressing recombinant VZV gE protein were collected, sonicated, and centrifuged at 12000 r / min for 15 min. Cell debris was discarded, and the supernatant was placed in a dialysis bag (Yuanye Biotechnology SP131378) and dialyzed for 12 h. The dialysate was 10 g NaHCO3 + 186.6 mg EDTA·2Na + 500 ml distilled water. The protein solution obtained from dialysis was filtered through Ni... 2+ -NTA metal chelate protein purification column (Detai Biotechnology) was used to purify VZV gE recombinant protein. Results are as follows: Figure 4 As shown, the purified recombinant protein was subjected to SDS-PAGE, and a specific target band of 59 kDa was visible, with a purity of approximately 90%.

[0093] Example 2. Preparation of reagents for detecting varicella-zoster virus (VZV) infection

[0094] S1. Preparation of streptavidin magnetic beads

[0095] (1) Activation of carboxyl magnetic beads

[0096] Take the stock solution of carboxylated magnetic beads (100 mg / ml, particle size 1 μm), gently shake to mix, take 100 μl of the stock solution, add 1 ml of 0.01 M phosphate buffer (pH 7.4), gently shake to wash, and place on a magnetic rack for recovery. Repeat this step three times, separating the magnetic beads and discarding the supernatant.

[0097] Add 200 μl of freshly prepared 5 mg / ml NHS (N-hydroxysuccinimide) and 200 μl of freshly prepared 5 mg / ml EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) solution to the magnetic beads, and incubate at 37 °C for 2 h to activate the magnetic beads. Then, sort the magnetic beads for 2 min and wash them three times with 1 ml of 0.01 M phosphate buffer (pH 7.4) to remove the activator, obtaining activated carboxyl magnetic beads.

[0098] (2) Covalent coupling of carboxyl magnetic beads and streptavidin

[0099] Activated carboxyl magnetic beads were placed in 100 μL of coupling buffer containing 1 mg / mL streptavidin [10 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl), 0.02 mol / L MES, pH 5.0] and gently stirred at 4 °C for 12 h. The magnetic beads were then separated and the supernatant was removed. The magnetic beads were washed three times with 1 mL of 0.01 M phosphate buffer (pH 7.4), and then resuspended in 500 μL of 0.01 M phosphate buffer (pH 7.4).

[0100] (3) Peeling and re-coupling of physically attached streptavidin

[0101] Streptavidin-conjugated magnetic beads were placed in a dialysis bag (Yuanye Biotechnology SP131414) for dialysis. The dialysis buffer was 500 ml of 0.01 M phosphate buffer (pH 7.4), and the buffer was changed every 4 hours. The device was magnetically stirred for 24 hours. After dialysis, the dialysis apparatus was placed in a 1.5 ml centrifuge tube and centrifuged at 1500 g for 1 min to obtain the dialyzed magnetic beads.

[0102] After dialysis, the magnetic beads were placed in 500 μl of coupling buffer containing 1 mg / mL streptavidin [10 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl), 0.02 mol / L MES, pH 5.0] and gently stirred at 4 °C for 12 h to fill the vacant carboxyl (-COOH) sites left by the removal of attached streptavidin. Then, the magnetic beads were washed three times with 1 mL of 0.01 M phosphate buffer (pH 7.4) to obtain the re-coupled streptavidin magnetic beads.

[0103] (4) Quenching and preservation of streptavidin magnetic beads

[0104] Add 500 μl of quenching buffer to the re-coupled streptavidin magnetic beads, mix gently, and incubate on a vortex mixer for 45 min. The quenching buffer was prepared by adding 10 ml of 0.01 M phosphate buffer (pH 7.4) to 0.638 ml of ethanolamine (2-hydroxyethylamine). Collect the magnetic beads using a magnetic rack and remove the supernatant. Store the magnetic beads in 20 mM phosphate buffer (pH 7.4) to obtain the streptavidin magnetic bead suspension.

[0105] (5) Preparation of working solution for streptavidin magnetic beads

[0106] The magnetic beads were removed from the streptavidin magnetic bead suspension obtained in step (4) and placed in the working solution to obtain the streptavidin magnetic bead working solution. The working solution was formulated as follows: it was prepared with 20 mM phosphate buffer (pH 7.4) and contained 0.3% (v / v) fetal bovine serum, 5 g / ml BSA (bovine serum albumin), 0.1 g / ml Lys (lysine), and 0.1% (v / v) Proclin-300 preservative.

[0107] S2. Preparation of biotinylated VZV gE recombinant protein

[0108] The concentration of the purified VZV gE recombinant protein from Example 1 was adjusted to 10 mg / ml using 20 mM phosphate buffer (pH 7.4). 2 mg of biotin was weighed and dissolved in 100 μl of purified water, and the mixture was thoroughly mixed to obtain a biotin solution. 10 μl of the 10 mg / ml VZV gE recombinant protein solution and 100 μl of the biotin solution were mixed and reacted at room temperature for 30 min. The resulting solution was dialyzed against 20 mM phosphate buffer (pH 7.4) to remove excess biotin, yielding a biotinylated VZV gE recombinant protein solution. An equal volume of glycerol was added, and the solution was stored at -20°C.

[0109] Example 3. Detection of varicella-zoster virus (VZV) infection

[0110] The positive samples used in the following experiments were serum samples from patients infected with VZV, and the negative samples were serum samples from healthy individuals not infected with VZV. All samples were obtained from Peking University International Hospital.

[0111] Preparation of acridine ester-labeled mouse anti-human IgM antibody solution: Using 0.05 mol / L NaHCO3 (pH 8.3) as buffer, mouse anti-human IgM antibody (Dening Biotechnology, DK251) was added at a mass ratio of 20:1 to acridine ester. After mixing, the mixture was reacted at room temperature in the dark for 1 hour. The mixture was then dialyzed against 10 mM Tris buffer (containing 0.05% Tween-20, pH 7.4) for 3 days to remove excess acridine ester. The labeled material was then removed, an equal volume of glycerol was added, and the mixture was stored at -20°C.

[0112] Preparation of activating solution A (an aqueous solution containing 0.1M HNO3 and 0.2% H2O2): Add HNO3 to purified water to a final concentration of 0.1M, and add H2O2 to a final concentration of 0.2%.

[0113] Preparation of activation solution B (an aqueous solution containing 0.25 mol / L NaOH and 0.1% CTAC): Add NaOH to purified water to a final concentration of 0.25 mol / L, and add CTAC (hexadecyltrimethylammonium chloride) to a final concentration of 0.1%.

[0114] 1. Comparison of different VZV gE recombinant proteins

[0115] The recombinant VZV gE protein expressed by HEK293F cells prepared in Example 1 (hereinafter referred to as "the protein of the present invention") was compared with the commercially available recombinant VZV gE protein expressed by Escherichia coli (hereinafter referred to as "the commercially available protein") (Abcamab43050).

[0116] Following the method described in Example 2, the protein of the present invention and the commercially available protein were biotinylated, and then 10 negative and 10 positive samples of the same type were tested. The detection method is as follows: 20 μl of 0.5 mg / ml streptavidin magnetic bead working solution obtained in S1(5) of Example 2, 100 μl of 1.0 μg / ml biotinylated (expressed by different hosts) VZV gE recombinant protein, and 10 μl of serum sample were mixed, reacted at 37°C for 10 min, washed 3 times, and then 100 μl of 0.05 mg / ml acridinium ester-labeled anti-human IgM antibody solution was added. After reacting at 37°C for 10 min and washing 3 times, 100 μl of excitation solution A and 100 μl of excitation solution B were added, and the luminescence value (RLU) was measured.

[0117] The results are shown in Tables 1 and 2. It can be seen that for the 10 negative samples, the detection signal background of the commercially available protein was higher than that of the protein of this invention, with stronger signals detected in the 3rd and 8th samples. This may be because the commercially available protein is a prokaryotic expression protein, and its spatial conformation differs somewhat from the native conformation of the VZV gE protein, allowing it to bind to non-specific substances in the samples. The protein of this invention showed lower detection values ​​for all 10 negative samples. For the 10 positive samples, the detection results of the commercially available protein showed strong signals in 7 samples, while the protein of this invention showed strong signals in 9 samples, with all signal intensities being stronger than those of the commercially available protein. This may be because the protein of this invention is expressed in human embryonic kidney cells, and its conformation is closer to that of the native gE protein of the human VZV virus, enabling it to more sensitively capture specific antibodies in the samples. Therefore, the protein of this invention has higher sensitivity and specificity compared to the commercially available protein.

[0118] Table 1. Detection results of 10 VZV-IgM negative samples using magnetic beads coated with different proteins.

[0119]

[0120] Table 2. Detection results of 10 VZV-IgM positive samples by magnetic beads coated with different proteins.

[0121]

[0122] 2. Comparison of different streptavidin-coupled magnetic bead methods

[0123] Five negative and five positive samples were tested using the streptavidin magnetic bead suspension obtained in S1(4) of Example 2 (hereinafter referred to as "streptavidin magnetic beads of the present invention") and the streptavidin magnetic bead suspension prepared by conventional methods (hereinafter referred to as "conventional streptavidin magnetic beads").

[0124] Preparation method of conventional streptavidin magnetic beads: Take the stock solution of carboxylated magnetic beads (100 mg / ml, particle size 1 μm), gently shake to mix, take 100 μl of the stock solution, add 1 ml of 0.01 M phosphate buffer (pH 7.4), gently shake to wash, and place on a magnetic rack for recovery. Repeat this step three times, and sort the magnetic beads to remove the supernatant. Add 200 μl of freshly prepared 5 mg / ml NHS and 200 μl of freshly prepared 5 mg / ml EDC solution, and incubate at 37°C for 2 hours. Wash the magnetic beads three times with 1 mL of 0.01 M phosphate buffer (pH 7.4) to remove the activator. Activated carboxyl magnetic beads were placed in 100 μl of coupling buffer containing 1 mg / mL streptavidin [10 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl), 0.02 mol / L MES, pH 5.0] and gently stirred at 4°C for 12 hours. The magnetic beads were then separated and the supernatant was discarded. The magnetic beads were washed three times with 1 mL of 0.01 M phosphate buffer (pH 7.4), and then resuspended in 500 μl of 0.01 M phosphate buffer (pH 7.4) to obtain the final product.

[0125] The detection method is as follows: Take 20 μl of 0.5 mg / ml (prepared by different methods) streptavidin magnetic bead suspension, 100 μl of 1.0 μg / ml biotinylated VZV gE recombinant protein prepared in Example 2, and 10 μl of serum sample, mix them, and react at 37℃ for 10 min; after washing 3 times, add 100 μl of 0.05 mg / ml acridinium ester-labeled anti-human IgM antibody solution, and react at 37℃ for 10 min; after washing 3 times, add 100 μl of excitation solution A and 100 μl of excitation solution B, and measure the luminescence value (RLU).

[0126] The results of sample testing using conventional streptavidin magnetic beads are shown in Table 3. The low detection value of positive samples indicates that the streptavidin covalent coupling efficiency of the magnetic beads is not high, and there is physical streptavidin adhering to the surface of the magnetic beads. During the repeated automatic cleaning process of the machine, the physically adhering streptavidin cannot stably bind to the magnetic beads and is peeled off from the magnetic beads. The high detection value of negative samples indicates the presence of non-specific adsorption.

[0127] Table 3. Results of routine streptavidin magnetic bead assay for VZV-IgM in negative and positive samples.

[0128] 1 2 3 4 5 negative sample 12667 13839 14021 12996 13876 positive sample 18900 19192 25438 27654 26543

[0129] The results of sample detection using the streptavidin magnetic beads of the present invention are shown in Table 4. The positive detection value was significantly improved, indicating that the magnetic beads have excellent sensitivity. This invention addresses the problem of low covalent coupling efficiency caused by the physical adsorption of streptavidin occupying episites. It achieves this by performing a first physical adsorption of streptavidin via dialysis and centrifugation, followed by re-coupling of streptavidin, which greatly improves the stable coupling efficiency.

[0130] Table 4. Results of streptavidin magnetic beads of the present invention in determining VZV-IgM negative and positive samples.

[0131] 1 2 3 4 5 negative sample 13769 14888 14828 16209 18775 positive sample 196706 161083 214037 210502 185035

[0132] 3. Verification of the effectiveness of the magnetic bead working fluid

[0133] Five negative and five positive samples were tested using the streptavidin magnetic bead suspension obtained in S1(4) of Example 2 and the streptavidin magnetic bead working solution obtained in S1(5) of Example 2, respectively.

[0134] The detection method is as follows: Take 20 μl of 0.5 mg / ml streptavidin magnetic bead suspension / streptavidin magnetic bead working solution, 100 μl of 1.0 μg / ml biotinylated VZV gE recombinant protein prepared in Example 2, and 10 μl of serum sample. After mixing, react at 37℃ for 10 min and wash 3 times. Then add 100 μl of 0.05 mg / ml acridinium ester-labeled anti-human IgM antibody solution, react at 37℃ for 10 min and wash 3 times. Then add 100 μl of excitation solution A and 100 μl of excitation solution B, and measure the luminescence value (RLU).

[0135] The results are shown in Tables 5 and 6. The working solution provided by this invention can effectively block vacant epitopes on streptavidin magnetic beads, resulting in a significant decrease in the detection value of negative samples, and has extremely high specificity.

[0136] Table 5. Results of streptavidin magnetic bead working solution assay for VZV-IgM in negative and positive samples.

[0137] 1 2 3 4 5 negative sample 3976 4576 4387 3432 5342 positive sample 200006 173862 204878 212574 199773

[0138] Table 6. Results of streptavidin magnetic bead suspension assay for VZV-IgM in negative and positive samples.

[0139] 1 2 3 4 5 negative sample 12669 13889 14828 17906 21876 positive sample 180432 161876 213436 220201 185043

[0140] 4. Determination of the working concentration of streptavidin magnetic beads

[0141] Working solutions of streptavidin magnetic beads with concentrations of 1.0 mg / ml, 0.5 mg / ml, and 0.25 mg / ml were prepared according to the method in Example 2. One VZV-IgM negative sample and one VZV-IgM positive sample were tested, with each sample tested three times.

[0142] The detection method is as follows: Take 20 μl (different concentrations) of streptavidin magnetic bead working solution, 100 μl of 1.0 μg / ml biotinylated VZV gE recombinant protein prepared in Example 2, and 10 μl of serum sample. After mixing, react at 37℃ for 10 min and wash 3 times. Then add 100 μl of 0.05 mg / ml acridinium ester-labeled anti-human IgM antibody solution, react at 37℃ for 10 min and wash 3 times. Then add 100 μl of excitation solution A and 100 μl of excitation solution B, and measure the luminescence value (RLU).

[0143] The results are shown in Table 7. When the concentration of streptavidin magnetic beads was 0.5 mg / ml, RLU 阳性 / RLU 阴性 Therefore, the optimal working concentration of streptavidin magnetic beads is 0.5 mg / ml.

[0144] Table 7. Optimal working concentration of streptavidin magnetic beads

[0145] Streptavidin magnetic bead concentration 1.0mg / ml 0.5mg / ml 0.25mg / ml <![CDATA[RLU 阴性 (n=3)]]> 4024 2375 1428 <![CDATA[RLU 阳性 (n=3)]]> 191413 167276 67149 <![CDATA[RLU 阳性 / RLU 阴性 ]]> 47.6 70.4 47.0

[0146] 5. Determination of the thermal stability of streptavidin magnetic beads

[0147] A 0.5 mg / ml streptavidin magnetic bead working solution was prepared according to the method in Example 2, and then stored at 2-8℃ (15 days) and 37℃ (3 days, 6 days, 9 days), respectively. The streptavidin magnetic bead working solutions treated at different temperatures were used to detect one negative sample and one positive sample, with each sample tested three times. The detection efficacy of streptavidin magnetic beads on VZV-IgM negative and positive samples under different storage conditions was compared.

[0148] The detection method is as follows: Take 20 μl of streptavidin magnetic bead working solution (treated at different temperatures), 100 μl of biotinylated VZV gE recombinant protein prepared in Example 2 at 1.0 μg / ml, and 10 μl of serum sample. After mixing, react at 37℃ for 10 min and wash 3 times. Then add 100 μl of acridinium ester-labeled anti-human IgM antibody solution, react at 37℃ for 10 min and wash 3 times. Then add 100 μl of excitation solution A and 100 μl of excitation solution B, and measure the luminescence value (RLU).

[0149] The results are shown in Table 8. The RLU values ​​of the streptavidin magnetic bead working solution after being placed at 37℃ for 3-9 days were basically the same as those of the samples after being placed at 2-8℃, with no significant difference, indicating that the streptavidin magnetic beads prepared in this invention have high stability.

[0150] Table 8. Results of thermal stability test of streptavidin magnetic beads

[0151]

[0152] Example 4. Preparation of a kit for detecting varicella-zoster virus (VZV) infection

[0153] 1. Components of the reagent kit

[0154] R1 reagent: Streptavidin magnetic bead working solution (0.5 mg / ml);

[0155] R2 reagent: Biotinylated VZV gE recombinant protein solution (1.0 μg / ml);

[0156] R3 reagent: Acridinium ester-labeled mouse anti-human IgM antibody solution (0.05 mg / ml);

[0157] Activation solution A: An aqueous solution containing 0.1M HNO3 and 0.2% H2O2;

[0158] Activation solution B: An aqueous solution containing 0.25 mol / L NaOH and 0.1% CTAC;

[0159] Product manual.

[0160] 2. Preparation of the reagent kit

[0161] (1) Prepare streptavidin magnetic bead working solution (R1 reagent) using the same method as in Example 2 above. Under aseptic conditions, fill the reagent bottle with R1 reagent and label it.

[0162] (2) Prepare biotinylated VZV gE recombinant protein solution (R2 reagent) using the same method as in Example 2 above. Under sterile conditions, fill the reagent bottle with R2 reagent and label it.

[0163] (3) Prepare acridil ester-labeled mouse anti-human IgM antibody solution (R3 reagent) using the same method as in Example 3 above. Under sterile conditions, fill the R3 reagent bottle and label it.

[0164] (4) Prepare activation solution A and activation solution B using the same method as in Example 3 above. Under sterile conditions, put activation solution A and activation solution B into reagent bottles and label them respectively.

[0165] (5) Put the reagent bottles containing reagent R1, reagent R2, reagent R3, activation solution A, and activation solution B, along with the product instructions, into the kit and affix a label to the kit shell.

[0166] 3. Instructions for use of the reagent kit

[0167] The steps for detecting VZV infection using the above kit are as follows: Take 20 μl of R1 reagent, 100 μl of R2 reagent and 10 μl of blood sample (serum or plasma), mix them, and react at 37℃ for 10 min; after washing 3 times, add 100 μl of R3 reagent and react at 37℃ for 10 min; after washing 3 times, add 100 μl of excitation solution A and 100 μl of excitation solution B, and measure the luminescence value (RLU).

[0168] Result interpretation: When RLU is greater than or equal to 100,000, it is judged as positive (infected with VZV); when RLU is less than 100,000, it is judged as negative (not infected with VZV).

Claims

1. A reagent for detecting varicella-zoster virus infection, characterized in that, Including reagents R1 and R2; The R1 reagent is a streptavidin magnetic bead working solution, which is prepared by: first coupling activated carboxyl magnetic beads with streptavidin; dialyzing the magnetic beads obtained from the first coupling to remove streptavidin physically bound to the magnetic beads; second coupling the dialyzed magnetic beads with streptavidin; quenching the magnetic beads obtained from the second coupling, and then placing them in the working solution; the working solution is an 18-22 mM phosphate buffer solution with pH 7.2-7.6 containing 0.2%-0.4% (v / v) fetal bovine serum, 4-6 g / ml bovine serum albumin and 0.1-0.3 g / ml lysine. The R2 reagent is a biotinylated VZV gE recombinant protein, which is an extracellular domain fragment of the varicella-zoster virus gE glycoprotein expressed by human embryonic kidney cells, as shown in SEQ ID NO:

1.

2. The reagent according to claim 1, characterized in that, The magnetic beads obtained from the first coupling were dialyzed using a 0.01-0.03 M phosphate buffer solution at pH 7.2-7.6; and / or the magnetic beads obtained from the second coupling were quenched using a quenching buffer solution composed of 2-hydroxyethylamine and a 0.01-0.03 M phosphate buffer solution at pH 7.2-7.

6.

3. The reagent according to claim 1, characterized in that, The human embryonic kidney cells were HEK293F cells.

4. The reagent according to claim 1, characterized in that, It also includes R3 reagent; said R3 reagent is an acridinium ester-labeled anti-human IgM antibody.

5. A method for preparing the reagent for detecting varicella-zoster virus infection according to any one of claims 1 to 4, characterized in that, include: S1: The activated carboxyl magnetic beads are coupled with streptavidin for the first time; The magnetic beads obtained from the first coupling were dialyzed to remove streptavidin physically bound to the beads; the dialyzed magnetic beads were then coupled with streptavidin a second time; the magnetic beads obtained from the second coupling were quenched and then placed in a working solution to obtain a streptavidin magnetic bead working solution; the working solution was a 18-22 mM phosphate buffer solution with pH 7.2-7.6 containing 0.2%-0.4% (v / v) fetal bovine serum, 4-6 g / ml bovine serum albumin and 0.1-0.3 g / ml lysine. S2: The extracellular domain fragment of varicella-zoster virus gE glycoprotein with the amino acid sequence shown in SEQ ID NO: 1 was expressed using human embryonic kidney cells, and the fragment was labeled with biotin to obtain biotinylated VZV gE recombinant protein.

6. The method according to claim 5, characterized in that, The magnetic beads obtained from the first coupling were dialyzed using a 0.01-0.03 M phosphate buffer solution at pH 7.2-7.6; and / or the magnetic beads obtained from the second coupling were quenched using a quenching buffer solution composed of 2-hydroxyethylamine and a 0.01-0.03 M phosphate buffer solution at pH 7.2-7.

6.

7. The method according to claim 5, characterized in that, The human embryonic kidney cells were HEK293F cells.

8. A kit comprising the reagent for detecting varicella-zoster virus infection as described in any one of claims 1 to 4.

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