A kind of feline parvovirus antibody fluorescent microsphere detection test paper and preparation method thereof

Abstract

The present invention provides a fluorescent microsphere test strip for feline parvovirus antibody and a preparation method thereof. Based on lateral flow immunoassay, Staphylococcus A protein is combined with Eu (III) chelated microparticles, and VP2 protein captures FPV antibodies at the same time. The more antibodies there are within the tolerance range of the test strip, the higher the fluorescence value that can be detected by the instrument, thereby realizing rapid quantitative indirect detection of FPV antibodies in cat serum, overcoming the defects of poor stability, photobleaching, and low sensitivity of traditional fluorescent markers. At the same time, the test strip of the present invention is extremely simple to operate, and the entire detection process only takes 20 to 30 minutes, which can be easily carried out on site and no longer relies on complex laboratory equipment and procedures. By testing cat serum samples and clinical samples, the detection limit of the fluorescent microsphere test strip is evaluated, and it is verified that it has high sensitivity, specificity, accuracy, repeatability and stability for FPV antibodies.

Description

Technical Field

[0001] The invention relates to the field of antibody detection test strips, and in particular to a feline parvovirus antibody fluorescent microsphere detection test strip and a preparation method thereof. Background Art

[0002] Feline panleukopenia virus (FPV) is a Parvoviridae virus that can cause panleukopenia syndrome in felines. Cats infected with FPV will show typical clinical symptoms, including high fever, loss of appetite, vomiting, diarrhea, weight loss, weakness, leukopenia, etc. FPV infection can cause immunosuppression in cats, and is often accompanied by secondary infection with other pathogens. Currently, regular vaccination is used to prevent the disease, which can significantly improve the resistance of cats to the disease. Therefore, detecting FPV antibody levels has important application value in protecting cats.

[0003] Vaccines protect cats from severe disease by inducing humoral and cellular immunity. For FPV infection, antibody titers are correlated with protective efficacy. FPV protective antibody levels can be determined in diagnostic laboratories by virus neutralization tests (VN) or hemagglutination inhibition (HI) tests. Although the HI test is considered the gold standard for FPV antibody detection, there is still no precise detection standard. Generally speaking, the FPV protective HI antibody titer threshold is 1:20 to 1:40, and greater than or equal to 1:40 is considered to prevent FPV, while the FPV protective serum neutralizing (SN) test antibody titer threshold is 1:10 to 1:32.

[0004] At present, the enzyme-linked immunosorbent assay (ELISA) is still mainly used clinically to detect FPV antibodies. Although ELISA can produce quantitative results with high sensitivity and good specificity, there are still some inconveniences and shortcomings, such as complex operation process, relatively expensive, high operation cost, and cannot represent the protective titer of antibodies. It is unnecessary to vaccinate cats with serum antibody titers above the threshold. Therefore, it is imperative to establish a highly sensitive, specific and rapid detection method. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a feline parvovirus antibody fluorescent microsphere test strip and a preparation method thereof.

[0006] In the first aspect, the present invention first provides a feline parvovirus antibody fluorescent microsphere test strip, which is composed of a PVC bottom plate, a sample pad, a conjugation pad, an NC membrane and a water absorbent pad, characterized in that the NC membrane is sprayed with feline parvovirus VP2 protein and porcine IgG as a detection line and a quality control line, respectively, the embedding amount of feline parvovirus VP2 protein on the detection line is 1 mg / mL, and the embedding amount of porcine IgG on the quality control line is 0.25 mg / mL; SPA coupled with fluorescent microspheres is sprayed on the conjugation pad, the coupling mass ratio of SPA to fluorescent microspheres is 5:100, and the coupling buffer adopts pH=8.0, 20mM HEPES buffer.

[0007] Furthermore, the feline parvovirus VP2 protein is expressed using a CHO cell line that overexpresses the feline parvovirus VP2 gene.

[0008] Furthermore, the CHO cell line overexpressing the feline parvovirus VP2 gene is obtained by integrating the feline parvovirus VP2 gene into the CHO cell locus through the catalysis of transposase under the action of Tol2 transposon using the transposition system vector of the feline parvovirus VP2 gene.

[0009] Furthermore, the transposition system vector of the feline parvovirus VP2 gene is constructed by double-digesting the overexpression vector of the feline parvovirus VP2 gene with AseI and ClaI to obtain a CMV-EF1α-FPV VP2 tandem sequence, and then inserting the CMV-EF1α-FPV VP2 tandem sequence into a Tol2 vector.

[0010] Furthermore, the overexpression vector of the feline parvovirus VP2 gene contains CMV and EF1α dual promoter elements, wherein the nucleic acid sequence of the EF1α promoter is shown in SEQ ID NO.1, and the feline parvovirus VP2 gene is an optimized gene, and its nucleotide sequence is shown in SEQ ID NO.3.

[0011] Furthermore, the overexpression vector of the feline parvovirus VP2 gene is constructed by connecting the EF1α promoter and the optimized feline parvovirus VP2 gene fragment and then inserting the fragment into the multiple cloning site of the pIRES2-EGFP plasmid containing the CMV promoter.

[0012] Furthermore, the method of coupling the fluorescent microspheres with SPA includes:

[0013] (1) Washing of fluorescent microspheres: 10 mg / mL fluorescent microspheres were added to pure water, and centrifuged at 15,500 rpm, 10°C for 25 min for the first wash; the supernatant was removed, and the microspheres were resuspended in pure water, and centrifuged at 15,500 rpm, 10°C for 25 min for the second wash; the supernatant was removed, and the microspheres were resuspended in labeling buffer. If the microspheres aggregated after centrifugation, they were dispersed by ultrasound for 5 min; the labeling buffer was pH 5.3, 20 mM MES;

[0014] (2) Activation of fluorescent microspheres: Add 10 mg / ml EDC to the washed microspheres and mix quickly; then add 10 mg / ml NHS to the microspheres and mix quickly, sonicate at 20W for 5 min; incubate at 37°C with shaking for 30 min; centrifuge at 15500 rpm, 10°C for 25 min, remove the supernatant, and resuspend the microspheres in coupling buffer; the coupling buffer is pH=8.0, 20 mM HEPES; if the microspheres aggregate after centrifugation, disperse them by sonication for 5 min;

[0015] (3) Coupling of fluorescent microspheres with proteins: Take SPA, add activated fluorescent microspheres, mix quickly, and incubate at 37°C with shaking for 2 h; add BSA to a final concentration of 1%, mix quickly, and incubate at 37°C with shaking for 1 h; centrifuge the blocked microsphere-protein mixture at 15,500 rpm, 10°C for 25 min; remove the supernatant, resuspend the microspheres with a resuspension solution, and place at 4°C for later use; the resuspension solution is prepared using the following reagents in parts by weight: 0.12 parts of Tris, 5 parts of sucrose, 2.5 parts of trehalose, 1.5 parts of BSA, and 40.88 parts of water; the pH of the resuspension solution is 8.5; if the microspheres aggregate after centrifugation, disperse them by ultrasound for 5 min.

[0016] In a second aspect, the present invention also provides a method for preparing the feline parvovirus antibody fluorescent microsphere test strip described in any one of the above, comprising the following steps:

[0017] S1. Fluorescent microspheres coupled with SPA:

[0018] (1) Washing of fluorescent microspheres: 10 mg / mL fluorescent microspheres were added to pure water, and centrifuged at 15,500 rpm, 10°C for 25 min for the first wash; the supernatant was removed, and the microspheres were resuspended in pure water, and centrifuged at 15,500 rpm, 10°C for 25 min for the second wash; the supernatant was removed, and the microspheres were resuspended in labeling buffer. If the microspheres aggregated after centrifugation, they were dispersed by ultrasound for 5 min; the labeling buffer was pH 5.3, 20 mM MES;

[0019] (2) Activation of fluorescent microspheres: Add 10 mg / ml EDC to the washed microspheres and mix quickly; then add 10 mg / ml NHS to the microspheres and mix quickly, sonicate at 20W for 5 min; incubate at 37°C with shaking for 30 min; centrifuge at 15500 rpm, 10°C for 25 min, remove the supernatant, and resuspend the microspheres in coupling buffer; the coupling buffer is pH=8.0, 20 mM HEPES; if the microspheres aggregate after centrifugation, disperse them by sonication for 5 min;

[0020] (3) Coupling of fluorescent microspheres with proteins: Take SPA, add activated fluorescent microspheres, mix quickly, and incubate at 37°C for 2 h; add BSA to a final concentration of 1%, mix quickly, and incubate at 37°C for 1 h; centrifuge the blocked microsphere-protein mixture at 15,500 rpm, 10°C for 25 min; remove the supernatant, resuspend the microspheres with a resuspension solution, and place at 4°C for later use; the resuspension solution is prepared using the following reagents in parts by weight: 0.12 parts of Tris, 5 parts of sucrose, 2.5 parts of trehalose, 1.5 parts of BSA, and 40.88 parts of water; the pH of the resuspension solution is 8.5; if the microspheres aggregate after centrifugation, disperse them by ultrasound for 5 min;

[0021] S2. Assembly of fluorescent microsphere test strips:

[0022] (1) Preparation: The sample pad was saturated with buffer, air-dried for 2 hours, and stored in a sealed bag; the buffer was 20 mM Tris-HCl containing 1% Triton X-100, 0.3% BSA, and 0.5% casein at pH 8.0;

[0023] (2) Gold spraying: Set the parameters of the film spraying instrument to 10 μL / cm, spray the fluorescent microsphere-coupled SPA onto the conjugate pad, and dry it for 1 to 2 hours; after drying, place the conjugate pad in a sealed bag and store it for future use;

[0024] (3) NC membrane streaking: Set the parameters of the sprayer to 1 μL / cm, and spray feline parvovirus VP2 protein and porcine IgG on the NC membrane as the test line and quality control line, respectively, with a spacing of 3.5 mm between the two lines; dry the streaked NC membrane for 30 min; place the dried NC membrane in a sealed bag and store for later use;

[0025] (4) Test strip assembly and cutting: First, the NC membrane is attached to the middle of the PVC board, and the absorbent pad is attached to the C-line end of the PVC base plate. The edge of the absorbent pad is pressed on the NC membrane and overlapped with the NC membrane by 2 mm. The conjugate pad sprayed with fluorescent microsphere-coupled SPA antibodies is attached to the T-line end of the NC membrane. The edge of the conjugate pad is pressed on the NC membrane and overlapped with the NC membrane by 2 mm. Finally, the sample pad is attached to the other side of the conjugate pad. The edge of the sample pad is pressed on the conjugate pad and overlapped with the conjugate pad by 2 mm. The PVC base plate is cut into 3.9 mm wide test strips using a strip cutter and placed in a sealed bag for storage.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The feline parvovirus antibody fluorescent microsphere test strip provided by the present invention is based on lateral flow immunoassay (LFIA), SPA is combined with Eu (III) chelated microparticles, and VP2 protein is used to capture FPV antibodies. The more antibodies there are within the tolerance range of the test strip, the higher the fluorescence value that can be detected by the instrument, thereby realizing rapid quantitative indirect detection of FPV antibodies in cat serum. Unlike the traditional LFIA method, this method uses Eu (III) chelated microparticles as fluorescent reporters, overcoming the defects of poor stability, photobleaching, and low sensitivity of traditional fluorescent markers. At the same time, the operation of the fluorescent microsphere test strip is extremely simple, and the entire detection process only takes 20 to 30 minutes, so that the technology can be easily carried out on site, no longer relying on complex laboratory equipment and procedures. Through the testing of cat serum samples and clinical samples, the detection limit of the fluorescent microsphere test strip based on Eu (III) chelated microparticles was evaluated, and it was verified that it has high sensitivity, specificity, accuracy, repeatability and stability for FPV antibodies.

[0028] The fluorescent microsphere test strip of the present invention not only plays a key role in evaluating the FPV antibody level of cats to determine whether to vaccinate and evaluate new vaccines, but also has multiple functions. Its disease screening ability makes it a powerful tool for detecting whether cats are infected with FPV, and can provide important information even after vaccination. In addition, regular antibody level testing helps monitor the spread trend of FPV in cat populations, conduct virus monitoring and epidemic investigations. The test strip can also be used to evaluate treatment effects, help optimize vaccination strategies, and provide veterinarians with auxiliary diagnostic information, making it a comprehensive tool for maintaining the health of cat populations. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A schematic diagram of the pIRES2-EGFP plasmid structure provided in Example 1 of the present invention;

[0030] Figure 2 A schematic diagram of the structure of the overexpression vector of the dual promoter (CMV+EF1α)+FPV VP2 gene provided in Example 1 of the present invention;

[0031] Figure 3 The CMV+EF1α dual promoter FPV VP2 transposition system vector in Example 1 of the present invention;

[0032] Figure 4 The results of selecting different conjugates provided in Example 3 of the present invention;

[0033] Figure 5The selection results of different C-line coatings provided in Example 3 of the present invention;

[0034] Figure 6 The fluorescence detection trend change of the fluorescent microsphere test strip provided in Example 3 of the present invention;

[0035] Figure 7 The test results of different coupling buffers provided in Example 3 of the present invention;

[0036] Figure 8 This is the optimization result of the coupling amount of fluorescent microspheres and antibodies provided in Example 3 of the present invention;

[0037] Fig. 9 The T-line coating amount optimization result provided in Example 3 of the present invention;

[0038] Fig.10 The C-line coating amount optimization result provided in Example 3 of the present invention;

[0039] Fig.11 The serum hemagglutination inhibition titer-T / C fluorescence value calibration curve provided in Example 4 of the present invention;

[0040] Fig.12 The repeatability test results provided by Example 4 of the present invention;

[0041] Fig.13 The stability test results provided for Example 4 of the present invention. DETAILED DESCRIPTION

[0042] In order to further illustrate the present invention, the test paper and the preparation method thereof of the present invention are described in detail in combination with the examples below, but they should not be understood as limiting the scope of protection of the present invention. The feline parvovirus FPV-JT02 strain used in the present invention was isolated by this laboratory (providing the original source disclosure table) and stored in a -80°C refrigerator. Fluorescent microspheres (FCEU002, PS-COOH Europium Chelate Microspheres, europium chelate microspheres, excitation light: 340nm; release light: 620nm) are commercially available products. SPA antibodies were purchased from Sangon Biotech (Shanghai) Co., Ltd.; porcine IgG was purchased from Suzhou Biolon Technology Co., Ltd.

[0043] Example 1 Construction of FPVVP2 transposition system vector

[0044] 1. Optimization of feline parvovirus VP2 gene

[0045] After the genome of the FPV strain was extracted and sequenced, the VP2 gene sequence was obtained. The present invention optimized the codons according to the codon preference of Chinese hamster ovary cells, replaced the rare codons, reduced translation sequences, and released translation sequences with common codons, and adjusted the CG content and unfavorable peaks to extend the half-life of mRNA. The codon preference was adjusted from 0.66 to 0.93, and the CG content was adjusted from 35.16% to 56.13%. The VP2 gene sequence before optimization is shown in SEQ ID NO.2, and the VP2 gene sequence after optimization is shown in SEQ ID NO.3.

[0046] 2. Synthesis of EF1α promoter tandem with feline parvovirus VP2 gene

[0047] The EF1α promoter gene sequence was connected to the optimized VP2 gene sequence, and the EF1α promoter gene sequence was shown in SEQ ID NO. 1. The constructed sequence was sent to the company for gene synthesis (commissioned to Sangon Biotech (Shanghai) Co., Ltd.), namely EF1α-FPVVP2.

[0048] 3. Construction of FPVVP2 overexpression vector

[0049] The synthesized EF1α-FPV VP2 gene was cloned into the pIRES2-EGFP plasmid (commissioned to Sangon Biotech (Shanghai) Co., Ltd.) to obtain the FPV VP2 overexpression vector. The schematic diagram of the structure of the vector before and after cloning is shown in Figure 1 and Figure 2 shown.

[0050] 4. Construction of CMV+EF1α dual promoter FPVVP2 transposition system vector

[0051] The constructed FPV VP2 overexpression vector was double-digested with AseI and ClaI, and the excised fragment, namely CMV-EF1α-FPVVP2, was ligated into the transposition system vector pTol2 plasmid (purchased from Addgene) after double-digestion with AseI and ClaI using T4 ligase (purchased from Takara Biotechnology (Dalian) Co., Ltd.) to obtain the CMV+EF1α dual promoter FPV VP2 transposition system vector. The structural diagram of the vector is shown in Figure 3 shown.

[0052] Example 2 Construction of a CHO cell line overexpressing FPVVP2

[0053] CHO-S cells (purchased from Gibco) were cultured in DMEM / F12 medium containing 10% fetal bovine serum at 37°C and 5% CO2 until the logarithmic growth phase. 5The cells were inoculated into a 12-well cell culture plate for 24 hours and used for cell transfection when the cell confluence reached about 80%.

[0054] 50 μL of DMEM / F12 medium was added to 0.5 mL of serum-free medium (purchased from Henan Punuoyi Biological Products Research Institute Co., Ltd.), and then the CMV+EF1α dual promoter FPV VP2 transposition system vector (2.5 μg / well) constructed in Example 1 was transfected into the CHO-S cells obtained above under the action of transposase (purchased from Addgene) by liposome transfection to construct a CHO cell line overexpressing FPVVP2.

[0055] Liposome transfection method is as follows:

[0056] (1) Taking a 24-well plate as an example, take a 1.5 mL centrifuge tube and mark the vector name, add 50 μL of basic DMEM / F12 medium into the tube, then take 0.6 μg of the CMV+EF1α dual promoter transposon vector expressing FPVVP2 constructed in Example 4 and 0.2 μg of transposase, add them into the tube respectively, mix well, and let stand at room temperature for five minutes;

[0057] (2) Take another centrifuge tube, mark it as the transfection reagent tube, add 50 μL of basic DMEM / F12 medium into the tube, then add 2 μL of transfection reagent Lipofectamine3000 into the tube, mix thoroughly, and let stand at room temperature for five minutes;

[0058] (3) Add the mixed solution in step (2) dropwise to the plasmid tube in step (1), mix thoroughly, and let stand at room temperature for 25 min;

[0059] (4) Take out the cells for transfection, discard the old culture medium, wash each well twice with 1× PBS buffer, and then add 500 μL of new DMEM / F12 reduced serum medium;

[0060] (5) Add the mixed solution in step (3) dropwise into the wells, gently shake the culture plate to evenly distribute the mixed solution, and then place it in an incubator for culture. After incubation for 5 hours, remove the culture plate, replace with fresh complete culture medium and continue culturing; use the optimal G418 concentration for cell screening 48 hours after cell transfection. After drug treatment, the cells no longer die, count the cell suspension, inoculate 1 cell per well into a 96-well plate, and wait for cell proliferation. When the cell fusion rate in the 96-well plate reaches about 90%, expand the culture, and then proceed to the subsequent passaging and screening steps.

[0061] The CHO cells that stably overexpressed FPVVP2 with dual promoters were collected after transfection, and the expression level of FPV VP2 in each group was detected at the mRNA level by qPCR. The qPCR experimental process is as follows:

[0062] Primers for qPCR detection were designed for the target gene FPV VP2 and the internal reference gene β-actin. The CHO cell lines of the BC group, Ctrl-C group, Ctrl-E group, and OE group were collected and the total RNA of each group of cells was extracted with Trizol (Thermo Fisher Scientific), and then the total RNA was reverse transcribed into cDNA using a reverse transcription kit (purchased from Yubao Bioengineering (Dalian) Co., Ltd.). The cDNA was diluted at a ratio of 1:4 and used as a template. After qPCR detection, according to 2 -△△ The CT algorithm was used to calculate the relative expression of FPVVP2 mRNA.

[0063] The qPCR primers include FPV VP2-F and FPVVP2-R, the sequence of FPVVP2-F is shown in SEQ ID NO.4, specifically CTGAGAACAGGCGATGAGT; the sequence of FPV VP2-R is shown in SEQ ID NO.5, specifically TGGTGAGCGATGTAGGTGA.

[0064] The qPCR primers of the internal reference β-actin include β-actin-F and β-actin-R, the sequence of β-actin-F is such as SEQ ID NO.6, and the specific sequence is GTGCCCATTTATGAGGGCT; the sequence of β-actin-R is such as SEQ ID NO.7, and the specific sequence is AGCTGTGGTCACGAAGGAGT.

[0065] The total volume of the qPCR reaction system was 20.0 μL, including the following: 10.0 μL SYBR Green Mix, 2.0 μL diluted template, 1.0 μL each of primers (FPV VP2-F / FPV VP2-R), and 6.0 μL enzyme-free water.

[0066] The qPCR reaction conditions are: 95°C pre-denaturation for 30s, amplification phase (95°C for 10s, 55°C for 30s, 45 cycles), melting curve phase at 95°C for 15s, 60-95°C for 1min.

[0067] The results of qPCR experiments showed that the relative expression of FPVVP2 mRNA in CHO cell lines could reach more than 170. After purification by affinity chromatography, FPVVP2 protein with a purity of more than 95% could be obtained.

[0068] The overexpression vector of the dual promoter (CMV+EF1α) and FPV VP2 in series of the present invention can significantly increase the expression of FPV VP2 protein in CHO cell line. Compared with the traditional E. coli expression system, the production of inclusion bodies is avoided and the post-translation modification cannot be performed. The protein of CHO cell line is highly soluble and has higher protein activity, which is more convenient for subsequent separation and purification. At the same time, the present invention adopts a dual promoter vector, which can also significantly increase the expression of FPVVP2 protein in CHO cell line.

[0069] Example 3 Optimization of assembly conditions of fluorescent microsphere test strips

[0070] 1. Selection of fluorescent microsphere conjugates

[0071] Different conjugates, 70μg SPA and 70μg rabbit anti-cat antibody, were added to 1000μg activated microspheres to make test strips. 100μL of positive serum sample diluted 100 times with 10mM PBS buffer was added to each of the two fluorescent microsphere test strips. After 20 minutes, the T and C line fluorescence values ​​were detected respectively. The higher the fluorescence value, the better the coupling effect. The results are as follows Figure 4 As shown, it was found that SPA increased the T line fluorescence value and T / C value, while rabbit anti-cat antibody reduced the T line fluorescence value and T / C value, so SPA was used as the conjugate.

[0072] 2. Fluorescent microspheres and SPA coupling method

[0073] (1) Washing of fluorescent microspheres: Take 100 μL of microspheres (1 mg) and add them to 900 μL of pure water. Centrifuge at 15,500 rpm, 10°C for 25 min for the first wash. Remove the supernatant and resuspend the microspheres with 1000 μL of pure water. Centrifuge at 15,500 rpm, 10°C for 25 min for the second wash. Remove the supernatant and resuspend the microspheres with 1000 μL of labeling buffer (20 mM MES, pH = 5.3). If the microspheres aggregate after centrifugation, disperse them by ultrasonication for 5 min.

[0074] (2) Activation of fluorescent microspheres: EDC solution and NHS solution were prepared immediately. 25 μL EDC (10 mg / ml) was added to the washed microspheres and mixed quickly; then 25 μL NHS (10 mg / ml) was added to the microspheres and mixed quickly, and then ultrasonicated at 20 W for 5 min; incubated at 37°C for 30 min. Centrifuged at 15500 rpm, 10°C for 25 min, removed the supernatant, and resuspended the microspheres with 1000 μL coupling buffer (20 mM HEPES, pH = 8.0). If the microspheres aggregated after centrifugation, disperse them by ultrasonication for 5 min.

[0075] (3) Fluorescent microspheres coupled to proteins: Take 70 μg SPA, add 1000 μg activated microspheres, mix quickly, and incubate at 37°C for 2 h. Add BSA to a final concentration of 1%, mix quickly, and incubate at 37°C for 1 h. Centrifuge the blocked microsphere-antibody mixture at 15,500 rpm, 10°C for 25 min. Remove the supernatant, resuspend the microspheres with 1000 μL resuspension solution (Tris, 0.12 g; sucrose, 5 g; trehalose, 2.5 g; BSA, 1.5 g; water, 40.88 g, pH = 8.5), and place at 4°C for later use. If the microspheres aggregate after centrifugation, disperse them by ultrasound for 5 min.

[0076] 3. Selection of C-line coating

[0077] Use different C-line coatings to draw lines, pig IgG (0.5 mg / mL) and goat anti-rabbit antibody (0.5 mg / mL), use VP2 protein (1.3 mg / mL) to draw T lines, and make test strips. Add 100 μL of positive serum sample diluted 100 times with 10mM PBS buffer to these two fluorescent microsphere test strips, and detect the T and C line fluorescence values ​​after 20 minutes. The higher the fluorescence value, the better the effect of the C-line coating (such as Figure 5 ), pig IgG is the best C-line coating.

[0078] 4. Fluorescent microsphere test strip assembly method and determination of detection time

[0079] (1) Preparation: The sample pad was saturated with buffer, air-dried for 2 hours, and stored in a sealed bag; the buffer was 20 mM Tris-HCl containing 1% Triton X-100, 0.3% BSA, and 0.5% casein at pH 8.0;

[0080] (2) Gold spraying: Set the parameters of the film sprayer (10 μL / cm), spray the fluorescent microsphere-SPA complex on the conjugate pad, and place it in a drying oven to dry for 1 to 2 hours. After drying, place the conjugate pad in a sealed bag, mark it, and store it at room temperature for future use.

[0081] (3) NC membrane streaking: Set the parameters of the membrane sprayer (1 μL / cm), spray VP2 protein and porcine IgG on the NC membrane as the test line (T) and quality control line (C), respectively, with a spacing of 3.5 mm between the two lines; place the streaked NC membrane in an incubator to dry for 30 min. Place the dried binding pad in a sealed bag, mark it, and store it at room temperature for future use.

[0082] (4) Test strip assembly and cutting: The test strip is composed of a PVC base plate, a sample pad, an NC membrane, a binding pad and a water-absorbing pad. The specific steps for assembling the test strip are as follows: first, stick the NC membrane in the middle of the PVC board, stick the water-absorbing pad on the C line end of the PVC base plate, on the NC membrane and overlap with the NC membrane by 2 mm, stick the binding pad sprayed with fluorescent microspheres-SPA complex on the T line end, overlap with the NC membrane by 2 mm, and finally stick the sample pad, overlapping with the binding pad by 2 mm. Cut the attached PVC base plate into 3.9 mm wide test strips with a strip cutter, place them in a sealed bag, add desiccant to seal, mark them, and store them under dry conditions at room temperature.

[0083] 100 μL of serum sample diluted 100 times with 10 mM PBS buffer was dripped onto the sample pads of 5 test strips. The fluorescence value of each test strip was detected every minute to obtain the trend of the fluorescence value of the fluorescent microsphere test strip over time, the time required for the T / C value to reach the stable stage, and the duration (such as Figure 6 ). The T / C value fluctuated greatly before 20 minutes, was relatively stable during 20 to 25 minutes, and the T / C value of some test strips decreased after 25 minutes. In summary, it was chosen to measure the T / C value within 20 to 25 minutes after the sample was added.

[0084] 5. Selection of coupling buffer

[0085] Using different coupling buffers, Tris buffer (20mM Tris-HCl, pH=8.0) and HEPES (4-hydroxyethylpiperazineethanesulfonic acid) buffer (20mM HEPES, pH=8.0), SPA (5μg / 100μg fluorescent microspheres) was coupled and made into test strips, 100μL of positive serum sample diluted 100 times with 10mM PBS buffer (HI titer before dilution was measured to be 1:1024) was added to these two fluorescent microsphere test strips, and the T and C line fluorescence values ​​were detected after 20 minutes. The higher the fluorescence value, the better the coupling effect.

[0086] Test results such as Figure 7 As shown in the figure, the fluorescence value of C line in HEPES group is weaker than that in Tris group, but its T line and T / C value are higher than those in Tris group, so HEPES buffer is better than Tris buffer.

[0087] 6. Optimization of the amount of fluorescent microspheres and antibody conjugation

[0088] In the step ③ of coupling fluorescent microspheres with SPA antibodies, 10, 30, 50, and 70 μg of SPA were added to 1000 μg of activated microspheres (i.e., 1, 3, 5, and 7 μg / 100 μg of fluorescent microspheres) for coupling. 100 μL of positive serum samples diluted 100 times with 10 mM PBS buffer (HI titer before dilution was 1:1024) were added dropwise to 2 fluorescent microsphere test strips with different coupling amounts (1, 3, 5, and 7 μg / 100 μg of fluorescent microspheres), and the best coupling ratio was selected based on the fluorescence value obtained after 20 minutes of detection. The results are shown in Figure 2. Figure 8 As shown, 5 μg SPA / 100 μg fluorescent microspheres is the best coupling ratio.

[0089] 7. T-line coating optimization

[0090] Different concentrations of VP2 protein (0.7, 1.0, 1.3 mg / mL) were used to draw the T line, and pig IgG (0.5 mg / mL) was used to draw the C line. Eight test strips of each type were taken and divided into four groups. 100 μL of positive serum samples (HI titer before dilution was 1:1024) diluted 100, 1000, and 10000 times with 10 mM PBS buffer and negative serum were dripped onto each test strip, and the fluorescence values ​​of the T and C lines were detected after 20 minutes. The appropriate T line concentration was selected so that the T / C value had a good correlation with the hemagglutination inhibition titer.

[0091] The final HI titers of the test were 1:128, 1:16, 1:2 and negative samples. The T / C value was tested after 20 minutes. The results were as follows Fig. 9 As shown in the figure, when the T line embedding amount is 1.0 mg / mL, the T / C value has a good correlation with the serum hemagglutination inhibition titer. However, it was found that the T / C value was too small, which is not conducive to low titer detection, so the C line coating amount needs to be reduced. At the same time, the reason why the T / C value is chosen to quantify the antibody concentration is that, on the one hand, there is a positive relationship between the fluorescence intensity of the T line and the antibody concentration in the sample, and on the other hand, as an internal control to confirm whether the sample migrates to the detection line and reacts correctly, regardless of the antibody concentration in the sample, the fluorescence intensity of the C line remains relatively unchanged. In short, using the fluorescence value ratio of the T line to the C line as the test result can offset the intrinsic heterogeneity of the lateral flow test strip and the sample matrix, which is more reliable and reproducible than using only the T line for signal quantification.

[0092] 8. C-line coating optimization

[0093] VP2 protein (1.0 mg / mL) was used to draw the T line, and different concentrations of porcine IgG (0.5, 0.25, 0.1 mg / mL) were used to draw the C line. Eight test strips of each type were taken and divided into four groups. 100 μL of positive serum samples (HI titer before dilution was 1:1024) diluted 100, 1000, and 10000 times with 10 mM PBS buffer and negative serum were dripped on each test strip, and the T and C line fluorescence values ​​were detected after 20 minutes. Under the premise that the T / C value has a good correlation with the hemagglutination inhibition titer, the appropriate C line concentration was selected so that the positive T / C value is greater than 1 and the negative T / C value is less than 0.5.

[0094] The final HI titers of the test were 1:128, 1:16, 1:2 and negative samples. The T / C value was tested after 20 minutes. The results were as follows Fig.10 As shown, when the C-line embedding amount was adjusted to 0.25 mg / ml porcine IgG, the test strip detection had a good correlation with the serum hemagglutination inhibition titer, and the positive T / C value was greater than 1 and the negative value was less than 0.5.

[0095] Example 4 Fluorescent Microsphere Test Strip Performance Evaluation

[0096] 1. Sensitivity test

[0097] Use serial two-fold dilutions of FPV-positive serum (determined hemagglutination inhibition titer of 1:210), that is, the titers of diluted serum are 1:2 1 , 1:2 0 , 1:2 -1 , 1:2 -2 , 1:2 -3 , 1:2 -4 , 1:2 -5 , the sensitivity of the fluorescent microsphere test strip was evaluated, and each dilution was tested five times. The results are shown in Table 1. The minimum detection limit (LOD) of the fluorescent microsphere test strip for the HI titer of cat FPV antibody is 1:2 -5 .

[0098] Table 1 Sensitivity test data

[0099]

[0100]

[0101] 2. Establish a standard curve of serum hemagglutination inhibition titer-T / C fluorescence value

[0102] The equation: log(agonist) vs. response - a variable slope (four parameters) was used to fit the data to more accurately describe the relationship between the T / C value of each sample and its hemagglutination inhibition titer, and a serum hemagglutination inhibition titer-T / C fluorescence value calibration curve was constructed. Fig.11 As shown. Using log(agonist) vs. response - a variable slope (four-parameter) model was used to determine the standard curve of antibody titer (Y = Bottom + (Top-Bottom) / (1 + 10^((LogEC50-X)*HillSlope)), R 2 =0.9941, Bottom=-0.1938, Top=1.771, LogEC50=-3.132, HillSlope=0.2573). The effective detection range of the fluorescent microsphere test strip, i.e., the limit of quantitation (LOQ), is 1:2 -4 ~1:2 0 .

[0103] 3. Specificity test

[0104] The specificity of the fluorescent microsphere test strips was evaluated by testing serum samples containing antibodies with positive neutralization titers against closely related feline viruses (such as feline herpes virus and feline calicivirus) using the prepared fluorescent microsphere test strips. The specificity experiment of the fluorescent microsphere test strips was carried out using feline herpes virus and feline calicivirus serum. The results showed that the fluorescent microsphere test strips could detect FPV-positive serum and had no cross-reactivity with other serums. The T / C values ​​of the test results of other virus antibody-positive serum were similar to those of negative serum.

[0105] 4. Repeatability test

[0106] In order to evaluate the intra-batch and inter-batch reproducibility of fluorescent microsphere test strips, three batches of fluorescent microsphere test strips were prepared and used to measure serum samples with different HI titers (1:2 -1 , 1:2 -2 , 1:2 -3 Each serum sample was measured five times, and the results were as follows: Fig.12 The intra-assay and inter-assay CV values ​​were both lower than 10% (as shown in Table 2), meeting the precision requirements.

[0107] Table 2 Repeatability test data

[0108]

[0109]

[0110] 5. Clinical sample testing

[0111] 84 clinical samples from Changchun City, Jilin Province, China were analyzed. Clinical serum samples were diluted 100 times using 10mM PBS buffer. Subsequently, 100μL of the diluted sample was dripped onto the sample pad of the fluorescent microsphere test strip. After 20 minutes, the fluorescence intensity at the T line and C line of the test strip was measured using a dry immunofluorescence analyzer for quantitative analysis. At the same time, these samples were subjected to HI test for comparative evaluation. The hemagglutination test and hemagglutination inhibition test refer to the national standards of the People's Republic of China (GB / T14926.53 and GB / T 14926.54).

[0112] The results are shown in Table 3. Of the 84 clinical samples tested, 66 were identified as FPV antibody positive and 18 samples were determined to be negative. A total of 66 FPV antibody positive samples and 18 negative samples were detected by the coagulation inhibition test.

[0113] Table 3 Comparison of fluorescent microsphere test strips and HI detection

[0114]

[0115] 6. Stability test

[0116] The assembled fluorescent microsphere test strips and desiccant were sealed in an aluminum foil bag and stored at 25°C for 0, 1, and 3 months using diluted serum with known HI titer (1:2 0 , 1:2 -1 , 1:2 -2 , 1:2 -3 , 1:2 -4 ) to evaluate the T / C value and conduct stability tests on the fluorescent microsphere test strips. Fig.13 As shown, the CV values ​​are all less than 15%.

[0117] Through the above research, the present invention has developed a simple and rapid fluorescent microsphere test strip, which is based on the lateral flow immunoassay (LFIA) method and combined with Eu (III) chelated microparticles to quantitatively determine the FPV antibody titer in serum. The specificity of the fluorescent microsphere test strip depends on the FPV VP2 protein coated on the T line and the SPA coupled to the fluorescent microspheres. In this study, we constructed the FPV-VP2-pCold expression plasmid. We added 6 histidine sequences to the C-terminus of the VP2 fragment to facilitate protein detection and purification. At the same time, based on the cold shock system, the VP2 protein was expressed in a soluble form. Low temperature induction is conducive to the correct folding of the protein and reduces the formation of inclusion bodies. By optimizing the culture conditions, we found that at 15°C, 0.4mMIPTG induced for 16 hours, the expression of soluble VP2 protein was the highest. During the purification process, in order to maintain the solubility and activity of the recombinant protein, protein aggregation, denaturation and inactivation should be avoided as much as possible. The pH value of the binding buffer and elution buffer is crucial to maintaining the solubility and stability of the recombinant protein. In this study, the pI value of VP2 protein was calculated based on the amino acid sequence of the recombinant protein (pI = 5.44), so the binding buffer and elution buffer with pH = 8.0 were selected for separation and purification. Under the optimal culture and purification conditions, VP2 protein was isolated by Ni-NTA affinity chromatography. Through detection, the total yield of VP2 per liter of bacterial culture fluid was about 1.319 mg.

[0118] When selecting the test strip preparation conditions, we first compared two conjugates, namely SPA and rabbit anti-cat antibody. The comparison found that SPA increased the fluorescence value and T / C value of the T line, while rabbit anti-cat antibody reduced the fluorescence value and T / C value of the T line. This is because each domain of SPA can recognize and bind to the specific amino acid sequence of the IgG Fc region, so that SPA can firmly bind to IgG, thereby binding more FPV VP2 antigens. Therefore, we chose SPA as the best conjugate. Secondly, different C-line coatings were selected for streaking, pig IgG and goat anti-rabbit antibody. The results showed that pig IgG increased the fluorescence value of the C line, and goat anti-rabbit antibody reduced the fluorescence value of the C line. Therefore, we selected pig IgG as the best C-line coating. We compared the effects of two different coupling buffers, Tris and HEPES buffer, on the coupling effect of fluorescent microspheres and SPA. After comparison, it was found that HEPES buffer can increase the fluorescence value and T / C value of the T line, while Tris buffer reduces the fluorescence value and T / C value of the T line, but increases the fluorescence value of the C line. This may be because HEPES buffer can maintain the stability and activity of fluorescent microspheres, while Tris buffer may be less suitable for the combination of fluorescent microspheres and SPA, resulting in the T-line fluorescence value and T / C value that should be combined with more fluorescent microspheres when using Tris buffer for coupling, which did not reach the height when using HEPES buffer. Therefore, we chose HEPES buffer as the best coupling buffer. Next, we explored the effect of different coupling amounts on the fluorescent microsphere test strips and found that when 5μg SPA was coupled with 100μg fluorescent microspheres, the T-line fluorescence value and T / C value reached the highest, while when the coupling amount was too high or too low, the T-line fluorescence value and T / C value both decreased. On the one hand, when SPA is too little, the SPA on the fluorescent microspheres is insufficient and cannot effectively bind to the FPVVP2 antigen, while when the coupling amount is too high, the SPA on the fluorescent microspheres is too much, resulting in fluorescence quenching of the fluorescent microspheres or aggregation of the fluorescent microspheres, affecting the output of the fluorescent signal. Therefore, we chose 5μg SPA / 100μg fluorescent microspheres as the best coupling amount. Finally, we optimized the embedding amount of the T and C lines of the test strip and found that when the embedding amount of the T line was 1.0 mg / mL VP2 protein and the embedding amount of the C line was 0.25 mg / mL porcine IgG, the T / C value of the test strip was best correlated with the serum HI titer, and the positive T / C value was greater than 1, and the negative T / C value was less than 0.5, with a better interpretation effect. This may be because the embedding amount of the T line and C line can affect the capture efficiency of the fluorescent microspheres and the intensity of the background signal. Too high or too low an embedding amount will reduce the sensitivity and specificity of the test strip. Therefore, we chose 1.0 mg / mL VP2 protein and 0.25 mg / mL porcine IgG as the optimal embedding amount.

[0119] The test using this test strip is based on a direct competitive immunoassay performed on a lateral flow test strip with a detection time of approximately 20 minutes and a limit of detection (LOD) of 1:2 for the HI titer of anti-FPV in cat serum. -5 , detection range (LOQ) is 1:2 -4 ~1:2 0 , indicating that the fluorescent microsphere test strip has a faster reaction speed and higher sensitivity, and can detect the presence of FPVVP2 antibodies in a short time. The dilution of serum with PBS buffer can cover the FPV antibody HI titer range in healthy cat serum and the high antibody level caused by infection or immunity. The coefficient of variation (CV) within and between batches of the test strips in the test was less than 15%, and the repeatability and stability were satisfactory.

[0120] In addition, the results of clinical experiments showed that the test results of the fluorescent microsphere test strips were basically consistent with the traditional standard method, proving its reliability and effectiveness in monitoring FPV antibodies in cats on site. The test strips will provide detection value for clinical applications. Finally, we conducted a stability test on the test strips, storing the test strips at 25°C for 0, 1, and 3 months, and using serum with different HI titers for testing. The results showed that the CV values ​​of the test strips were all less than 15%, indicating that the test strips did not have a significant performance degradation during storage and maintained good stability.

[0121] In summary, we have successfully developed an immunochromatographic test strip method based on Eu(III) chelated microparticles. It does not require expensive equipment, complicated operations and pre-treatment. It only needs to dilute the serum into sample buffer and drip it onto the test strip and wait for 20 minutes to quantitatively detect the FPV antibody level in cat serum. Due to its simplicity, high sensitivity and accuracy, it is very suitable as a primary clinical detection method, providing a feasible solution for the timely diagnosis and control of feline parvovirus infection, ensuring the health of cats.

[0122] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A feline parvovirus antibody fluorescent microsphere test strip, consisting of a PVC base plate, a sample pad, a conjugate pad, an NC membrane and a water absorbent pad, characterized in that: The NC membrane is sprayed with feline parvovirus VP2 protein and porcine IgG as a detection line and a quality control line, respectively, the embedding amount of feline parvovirus VP2 protein on the detection line is 1 mg / mL, and the embedding amount of porcine IgG on the quality control line is 0.25 mg / mL; Staphylococcus aureus protein A (SPA) coupled with fluorescent microspheres is sprayed on the conjugate pad, the coupling mass ratio of SPA to fluorescent microspheres is 5:100, and the coupling buffer adopts pH=8.0, 20mM HEPES buffer.

2. The feline parvovirus antibody fluorescent microsphere test strip according to claim 1, characterized in that: The feline parvovirus VP2 protein is expressed by a CHO cell line that overexpresses the feline parvovirus VP2 gene.

3. The feline parvovirus antibody fluorescent microsphere test strip according to claim 2, characterized in that, The CHO cell line overexpressing the feline parvovirus VP2 gene is obtained by integrating the feline parvovirus VP2 gene into the CHO cell gene locus through the catalysis of transposase by the transposition system vector of the feline parvovirus VP2 gene under the action of Tol2 transposon.

4. The feline parvovirus antibody fluorescent microsphere test strip according to claim 3, characterized in that: The transposition system vector of the feline parvovirus VP2 gene is constructed by double-digesting the overexpression vector of the feline parvovirus VP2 gene with AseI and ClaI to obtain a CMV-EF1α-FPV VP2 tandem sequence, and then inserting the CMV-EF1α-FPV VP2 tandem sequence into a Tol2 vector.

5. The feline parvovirus antibody fluorescent microsphere test strip according to claim 4, characterized in that: The overexpression vector of the feline parvovirus VP2 gene contains CMV and EF1α dual promoter elements, wherein the nucleic acid sequence of the EF1α promoter is shown in SEQ ID NO.1, and the feline parvovirus VP2 gene is an optimized gene, and its nucleotide sequence is shown in SEQ ID NO.

3.

6. The feline parvovirus antibody fluorescent microsphere test strip according to claim 5, characterized in that: The overexpression vector of the feline parvovirus VP2 gene is constructed by connecting the EF1α promoter and the optimized feline parvovirus VP2 gene fragment and then inserting the fragment into the multiple cloning site of the pIRES2-EGFP plasmid containing the CMV promoter.

7. The feline parvovirus antibody fluorescent microsphere test strip according to claim 1, characterized in that: Methods for coupling fluorescent microspheres to SPA include: (1) Washing of fluorescent microspheres: 10 mg / mL fluorescent microspheres were added to pure water, and centrifuged at 15,500 rpm, 10°C for 25 min for the first wash; the supernatant was removed, and the microspheres were resuspended in pure water, and centrifuged at 15,500 rpm, 10°C for 25 min for the second wash; the supernatant was removed, and the microspheres were resuspended in labeling buffer. If the microspheres aggregated after centrifugation, they were dispersed by ultrasound for 5 min; the labeling buffer was pH 5.3, 20 mM MES; (2) Activation of fluorescent microspheres: Add 10 mg / ml EDC to the washed microspheres and mix quickly; then add 10 mg / ml NHS to the microspheres and mix quickly, sonicate at 20W for 5 min; incubate at 37°C with shaking for 30 min; centrifuge at 15500 rpm, 10°C for 25 min, remove the supernatant, and resuspend the microspheres in coupling buffer; the coupling buffer is pH=8.0, 20 mM HEPES; if the microspheres aggregate after centrifugation, disperse them by sonication for 5 min; (3) Coupling of fluorescent microspheres with proteins: Take SPA, add activated fluorescent microspheres, mix quickly, and incubate at 37°C with shaking for 2 h; add bovine serum albumin (BSA) to a final concentration of 1%, mix quickly, and incubate at 37°C with shaking for 1 h; centrifuge the blocked microsphere-protein mixture at 15,500 rpm, 10°C for 25 min; remove the supernatant, resuspend the microspheres with a resuspension solution, and place at 4°C for later use; the resuspension solution is prepared using the following reagents in parts by weight: 0.12 parts of Tris, 5 parts of sucrose, 2.5 parts of trehalose, 1.5 parts of BSA, and 40.88 parts of water, and the pH of the resuspension solution is 8.5; if the microspheres aggregate after centrifugation, disperse them by ultrasound for 5 min.

8. A method for preparing a feline parvovirus antibody fluorescent microsphere test strip according to any one of claims 1 to 7, characterized in that: The following steps are involved: S1. Fluorescent microspheres coupled with SPA: (1) Washing of fluorescent microspheres: 10 mg / mL fluorescent microspheres were added to pure water, and centrifuged at 15,500 rpm, 10°C for 25 min for the first wash; the supernatant was removed, and the microspheres were resuspended in pure water, and centrifuged at 15,500 rpm, 10°C for 25 min for the second wash; the supernatant was removed, and the microspheres were resuspended in labeling buffer. If the microspheres aggregated after centrifugation, they were dispersed by ultrasound for 5 min; the labeling buffer was pH 5.3, 20 mM MES; (2) Activation of fluorescent microspheres: Add 10 mg / ml EDC to the washed microspheres and mix quickly; then add 10 mg / ml NHS to the microspheres and mix quickly, sonicate at 20W for 5 min; incubate at 37°C with shaking for 30 min; centrifuge at 15500 rpm, 10°C for 25 min, remove the supernatant, and resuspend the microspheres in coupling buffer; the coupling buffer is pH=8.0, 20 mM HEPES; if the microspheres aggregate after centrifugation, disperse them by sonication for 5 min; (3) Coupling of fluorescent microspheres with proteins: Take SPA, add activated fluorescent microspheres, mix quickly, and incubate at 37°C for 2 h; add BSA to a final concentration of 1%, mix quickly, and incubate at 37°C for 1 h; centrifuge the blocked microsphere-protein mixture at 15,500 rpm, 10°C for 25 min; remove the supernatant, resuspend the microspheres with a resuspension solution, and place at 4°C for later use; the resuspension solution is prepared using the following reagents in parts by weight: 0.12 parts of Tris, 5 parts of sucrose, 2.5 parts of trehalose, 1.5 parts of BSA, and 40.88 parts of water; the pH of the resuspension solution is 8.5; if the microspheres aggregate after centrifugation, disperse them by ultrasound for 5 min; S2. Assembly of fluorescent microsphere test strips: (1) Preparation: Saturate the sample pad with buffer, air-dry for 2 hours, and store in a sealed bag; The buffer was 20 mM Tris-HCl containing 1% Triton X-100, 0.3% BSA, and 0.5% casein, pH 8.0; (2) Gold spraying: Set the parameters of the film spraying instrument to 10 μL / cm, spray the fluorescent microsphere-coupled SPA onto the conjugate pad, and dry it for 1 to 2 hours; after drying, place the conjugate pad in a sealed bag and store it for future use; (3) NC membrane streaking: Set the parameters of the sprayer to 1 μL / cm, and spray feline parvovirus VP2 protein and porcine IgG on the NC membrane as the test line and quality control line, respectively, with a spacing of 3.5 mm between the two lines; dry the streaked NC membrane for 30 min; place the dried NC membrane in a sealed bag and store for later use; (4) Test strip assembly and cutting: First, the NC membrane is attached to the middle of the PVC board, and the absorbent pad is attached to the C-line end of the PVC base plate. The edge of the absorbent pad is pressed on the NC membrane and overlapped with the NC membrane by 2 mm. The conjugation pad sprayed with fluorescent microspheres coupled to SPA is attached to the T-line end of the NC membrane. The edge of the conjugation pad is pressed on the NC membrane and overlapped with the NC membrane by 2 mm. Finally, the sample pad is attached to the other side of the conjugation pad. The edge of the sample pad is pressed on the conjugation pad and overlapped with the conjugation pad by 2 mm. The PVC base plate is cut into 3.9 mm wide test strips using a strip cutter and placed in a sealed bag for storage.

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