Colloidal gold test strip for detecting cat gnRH antibody and preparation method and kit thereof

Abstract

The application discloses a colloidal gold test strip for detecting cat GnRH antibody and a preparation method and kit thereof. In the application, by spraying a colloidal gold-rabbit anti-cat IgG antibody conjugate on a combination pad, adsorbing a GnRH-C-BSA conjugate on a detection line, and adsorbing streptococcal protein G solution on a quality control line, a colloidal gold test strip capable of simply, quickly and efficiently detecting cat GnRH antibody is established. When the colloidal gold test strip is used for detecting cat GnRH antibody, the colloidal gold test strip has higher specificity, sensitivity and accuracy.

Description

Technical Field

[0001] This application relates to the field of immunochemical detection technology, and more specifically, to a colloidal gold test strip for detecting feline GnRH antibodies, its preparation method, and a reagent kit. Background Technology

[0002] Gonadotropin-releasing hormone (GnRH) is a neurohormone secreted by the hypothalamus and plays a crucial role in the regulation of vertebrate reproduction. Physiological doses of GnRH-1 can increase gonadotropin concentrations, promote the synthesis and secretion of gonadal hormones, stimulate follicle maturation and ovulation, or promote testicular development and sperm maturation, leading to and maintaining secondary sexual characteristics. Furthermore, GnRH-1 can directly affect the gonads, regulating the synthesis and secretion of gonadal steroid hormones and promoting gamete formation.

[0003] GnRH antibodies can induce immune castration, achieving a chemical castration effect by inhibiting sex hormone secretion. The principle is that antibodies produced by the body neutralize endogenous gonadotropin-releasing hormone (GnRH), thereby blocking the hypothalamic-pituitary-gonadal axis. After animals are injected with GnRH vaccines, they produce specific antibodies (GnRH antibodies). These antibodies bind to natural GnRH, preventing its interaction with GnRH receptors on the pituitary gland. Because GnRH signaling is blocked, the secretion of LH and FSH by the pituitary gland is significantly reduced. The decrease in LH and FSH suppresses the ability of the testes or ovaries to synthesize sex hormones (such as testosterone and estrogen), ultimately achieving functional castration.

[0004] In relevant applications, after animals are injected with GnRH vaccines, it is usually necessary to detect the expression level of GnRH antibodies to assess the castration effect of the GnRH vaccine. However, there is currently a lack of simple, rapid, efficient, and low-cost methods for detecting GnRH antibodies. Summary of the Invention

[0005] The main objective of this application is to provide a colloidal gold test strip for detecting feline GnRH antibodies, along with its preparation method and kit, to solve the technical problems of cumbersome operation, low efficiency, and high cost in existing GnRH antibody detection techniques, thereby achieving the technical effect of simple, rapid, efficient, and low-cost detection of GnRH antibodies.

[0006] To achieve the above objectives, a first aspect of this application provides a colloidal gold test strip for detecting feline GnRH antibodies. The colloidal gold test strip includes a PVC base plate, a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper. The sample pad, conjugate pad, nitrocellulose membrane, and absorbent paper are sequentially adhered to the PVC base plate and connected by overlapping. Detection lines and control lines are sequentially distributed on the nitrocellulose membrane along the direction from the conjugate pad to the absorbent paper.

[0007] The conjugate pad is coated with a colloidal gold-rabbit anti-cat IgG antibody conjugate solution; The detection line is adsorbed with a detection line solution, which includes a GnRH-C-BSA conjugate. The GnRH-C-BSA conjugate is a conjugate of a GnRH polypeptide with a cysteine ​​residue linked to its C-terminus and bovine serum albumin (BSA). The control line is adsorbed with control line solution, which includes streptococcal protein G.

[0008] In one optional embodiment, the amino acid sequence of VH-CDR1 of the rabbit anti-cat IgG antibody is shown in SEQ ID NO.1, the amino acid sequence of VH-CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of VH-CDR3 is shown in SEQ ID NO.3; The amino acid sequence of VL-CDR1 of the rabbit anti-cat IgG antibody is shown in SEQ ID NO.4, the amino acid sequence of VL-CDR2 is shown in SEQ ID NO.5, and the amino acid sequence of VL-CDR3 is shown in SEQ ID NO.6.

[0009] In one optional embodiment, in the colloidal gold-rabbit anti-cat IgG antibody conjugate, the weight ratio of the colloidal gold to the rabbit anti-cat IgG antibody is 400:(12~28). And / or, in terms of the concentration of colloidal gold, the concentration of the colloidal gold-rabbit anti-cat IgG antibody conjugate solution is 1~5 mg / mL; And / or, the amount of the colloidal gold-rabbit anti-cat IgG antibody conjugate solution sprayed onto the binding pad is 2~7 μL / cm.

[0010] In an optional embodiment, the amino acid sequence of the GnRH-C polypeptide in the GnRH-C-BSA conjugate is shown in SEQ ID NO.7; And / or, the molar ratio of the GnRH-C polypeptide to BSA is (8~12):1; And / or, the concentration of the GnRH-C-BSA conjugate in the detection line solution is 0.25~5 mg / mL; And / or, the amount of the detection line solution on the detection line is 0.5~1.5 μL / cm.

[0011] In one optional embodiment, the concentration of streptococcal protein G in the control solution is 0.25~1.5 mg / mL; And / or, the amount of the control line solution used for streaking on the control line is 0.5~1.5 μL / cm.

[0012] In one optional embodiment, along the long side of the colloidal gold test strip, the length of the sample pad is 10-15 mm, the length of the conjugation pad is 10-15 mm, the length of the nitrocellulose membrane is 20-30 mm, and the length of the absorbent paper is 12-20 mm. On the nitrocellulose membrane, the distance between the detection line and the conjugation pad is 10-15 mm; the distance between the detection line and the control line is 4-8 mm; and the distance between the control line and the absorbent paper is 4-8 mm.

[0013] In one optional embodiment, the preparation process of the colloidal gold-rabbit anti-cat IgG antibody conjugate includes: Adjust the pH of the colloidal gold solution to 4-10 and mix well for 5-30 min. Add rabbit anti-cat IgG antibody and react for 30-60 min. Then add blocking solution and block for 30-60 min. Separate the solid and liquid and take the solid.

[0014] In one optional embodiment, the preparation process of the GnRH-C-BSA conjugate includes: SMCC is reacted with BSA to obtain SMCC-BSA; The GnRH-C peptide is reacted with the SMCC-BSA to obtain the GnRH-C-BSA conjugate.

[0015] Secondly, this application provides a method for preparing the above-mentioned colloidal gold test strip, comprising the following steps: (1) A colloidal gold-rabbit anti-cat IgG antibody conjugate solution was sprayed onto a blank conjugate pad to obtain a conjugate pad; (2) Draw lines on the nitrocellulose membrane in sequence to adsorb the detection line solution and the quality control line solution, and obtain the nitrocellulose membrane after the lines are drawn; (3) The sample pad, conjugate pad, crisscrossed nitrocellulose membrane and absorbent paper are then pasted onto the PVC base plate in an overlapping manner.

[0016] Thirdly, this application provides a kit for detecting feline GnRH antibodies, the kit comprising the above-mentioned colloidal gold test strip.

[0017] The technical solutions provided by the embodiments of this application may include the following beneficial effects: In this application, a colloidal gold test strip for detecting feline GnRH antibodies was developed by spraying a colloidal gold-rabbit anti-cat IgG antibody conjugate onto the conjugate pad and adsorbing a GnRH-C-BSA conjugate onto the detection line. The colloidal gold test strip exhibits high specificity, sensitivity, and accuracy when used to detect feline GnRH antibodies.

[0018] In particular, the rabbit anti-cat IgG antibody used in this application can specifically bind to a variety of cat IgG antibodies, exhibiting high sensitivity and affinity, which is one of the reasons why the colloidal gold test strip of this application has high sensitivity. Attached Figure Description

[0019] The accompanying drawings, which form part of this application, are used to provide a further understanding of the application and to make other features, objects, and advantages of the application more apparent. The illustrative embodiments and descriptions of this application are used to explain the application and do not constitute an undue limitation of the application. In the drawings: Figure 1 The images show the detection results involved in the preparation of rabbit anti-cat IgG antibody in Example 1 of this application; wherein, (A) is the detection result of rabbit serum antibody titer in step 2 of Example 1; (B) is the flow cytometry sorting gating result in step 3 of Example 1; (C) is the result of antibody binding to cat IgG ELISA in step 4 of Example 1; and (D) is the SDS-PAGE image of 15 antibodies expressed in step 4 of Example 1. Figure 2 This is a diagram showing the binding effect of 15 rabbit anti-cat IgG monoclonal antibodies with the whole-cat monoclonal antibody in step 5 of Example 1 of this application; Figure 3 This is a graph showing the indirect immunofluorescence results of rabbit anti-cat IgG monoclonal antibody and cat IgG in step 6 of Example 1 of this application; Figure 4 This is a schematic diagram of the colloidal gold test strip in Embodiment 2 of this application; Figure 5 This is a graph showing the detection results of the colloidal gold test strip in Example 2 of this application; Figure 6 This is a graph showing the results of the dosage investigation of rabbit anti-cat IgG antibody mAb-38 in Example 3 of this application; Figure 7 This is a graph showing the results of investigating the coating concentration of the GnRH-C-BSA conjugate on a nitrocellulose membrane in Example 3 of this application; Figure 8 This is a diagram showing the results of the specificity investigation in Example 4 of this application; Figure 9 This is a graph showing the results of the sensitivity test in Example 4 of this application; Figure 10 This is a graph showing the results of the stability test in Example 4 of this application; Figure 11 The image shows the test results of the colloidal gold test strip in Comparative Example 1 of this application; Figure 12 This is a graph showing the test results of the colloidal gold test strip in Comparative Example 2 of this application. Detailed Implementation

[0020] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the scope of protection of the present application.

[0021] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this application are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate for the embodiments of this application described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0022] In this application, the terms "upper," "lower," "left," "right," "front," "rear," "top," "bottom," "inner," "outer," "middle," "vertical," "horizontal," "lateral," and "longitudinal" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. These terms are primarily for the purpose of better describing this application and its embodiments, and are not intended to limit the indicated device, element, or component to having a specific orientation, or to be constructed and operated in a specific orientation.

[0023] Furthermore, in addition to indicating location or positional relationship, some of the aforementioned terms may also have other meanings. For example, the term "above" may also be used in some cases to indicate a certain dependency or connection relationship. Those skilled in the art can understand the specific meaning of these terms in this application based on the specific circumstances.

[0024] Furthermore, the terms "installation," "setup," "equipped with," "connection," "linked," and "socketing" should be interpreted broadly. For example, "connection" can be a fixed connection, a detachable connection, or an integral structure; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, or an internal connection between two devices, components, or parts. Those skilled in the art can understand the specific meaning of the above terms in this application according to the specific circumstances.

[0025] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0026] The present invention will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed by the present invention.

[0027] Example 1: Preparation of rabbit anti-cat IgG antibody 1. Preparation of feline IgG Feline venous blood was collected, and serum was collected by centrifugation. The serum was diluted with 10-fold volume of PBS, and particulate matter was removed using a 0.45 μm filter. A Protein A gravity column was equilibrated with 4 column volumes of PBS. The filtered, diluted serum was added to the gravity column, and the column was washed with 4 column volumes of PBS. IgG antibodies were then eluted with 4 column volumes of 0.2 M glycine-HCl (pH 2.5). The eluent was collected, and 1 / 8 volume of Tris-HCl (pH 9.0) was added to adjust the pH. The solvent in the collected solution was replaced with PBS using a 30 kDa ultrafiltration tube to obtain purified feline IgG.

[0028] 2. Immunization of New Zealand rabbits with feline IgG Purified feline IgG was diluted to a concentration of 1 mg / mL and mixed with Freund's adjuvant at a ratio of 1:1 (V / V). The mixture was vortexed for 30 min and then used to immunize New Zealand rabbits. Immunization was repeated every 14 days for a total of 4 immunizations. Venous blood was collected before each immunization and after the last immunization to separate serum, which was then stored. Antibody titers were detected using ELISA. The results are as follows: Figure 1 As shown in Figure A, the serum antibody titer of the New Zealand rabbits reached 1:1093500 after the fourth immunization. The New Zealand rabbits were euthanized, and their spleens were collected for lymphocyte isolation.

[0029] 3. Cat IgG-specific B cell sorting and nested PCR 10 μg of BV421 fluorescent dye was incubated with cat IgG antibody at room temperature in the dark for 30 min for specific cell labeling. Pan B antibodies were then analyzed from the collected New Zealand rabbit spleen cells using flow cytometry. + IgM - IgG + and feline IgG + The live cells were sorted out, and the flow cytometry sorting results are as follows: Figure 1As shown in Figure B, single B cells were obtained, and a total of 96 single cells were selected and numbered mAb-(1~96). Using the Xinbei Biotechnology R202-02 reverse transcription kit, the RNA from the 96 selected rabbit spleen single cells was reverse transcribed into cDNA. For the cDNA of each single cell, two rounds of amplification were performed using KOD One™ PCR Master Mix reagent and primers 1~40 from the patent application (PCT / CN2024 / 120004) for both the heavy chain variable region (VH) and light chain variable region of the antibody gene. The amplification products were verified by 1.5% agarose gel electrophoresis. Pairs that simultaneously amplified the heavy chain variable region and the light chain variable region were selected for the next step of vector construction. Paired antibody genes were amplified in 84 of the 96 single cells.

[0030] 4. Antibody expression and ELISA The VH and VL genes of 84 antibody pairs were sequentially constructed into linearized pTT5 vectors linked with constant regions of the rabbit IgG heavy and light chains, and expressed in small quantities in the HEK293f eukaryotic expression system. After 3 days, expressing cells were collected, centrifuged at 4000 rpm for 20 minutes, and the supernatant was transferred to 96-well plates and stored at -20°C or used directly for ELISA detection.

[0031] ELISA test results as follows Figure 1 As shown in C, among the 84 monoclonal antibodies expressing low levels of expression, 15 monoclonal antibodies had OD values ​​of... 450 For values ​​greater than 1.0, the above 15 monoclonal antibodies were expressed in large quantities and analyzed by SDS-PAGE. The results are as follows: Figure 1 As shown in Figure D, clear target bands were visible in the 15 selected monomers near 50 and 25 kDa, with sizes consistent with expectations. Fifteen candidate rabbit anti-cat IgG monoclonal antibodies were obtained, corresponding to the numbers mAb-1, mAb-4, mAb-15, mAb-22, mAb-28, mAb-34, mAb-38, mAb-53, mAb-64, mAb-69, mAb-72, mAb-85, mAb-87, mAb-88, and mAb-94.

[0032] 5. EC of rabbit anti-cat IgG monoclonal antibody 50 Detection The binding affinity of the 15 rabbit anti-cat IgG monoclonal antibodies obtained above to the FPV whole-cat monoclonal antibody was determined by indirect ELISA. The FPV whole-cat monoclonal antibody was diluted with PBS to a concentration of 1 μg / mL, then added to an ELISA plate and incubated overnight at 4°C. The plate was washed and blocked the next day. Each rabbit anti-cat IgG monoclonal antibody expressed above was diluted to the following concentrations: 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, and 0.0001 μg / mL. Each dilution was added sequentially to the ELISA plate coated with the FPV whole-cat monoclonal antibody at a volume of 100 μL per well. After incubation, the plate was washed, and HRP-labeled goat anti-rabbit IgG was added as a secondary antibody for verification. Add TMB chromogenic solution to each well and react in the dark for 15 minutes. Then, terminate the chromogenic reaction with 1 M concentrated sulfuric acid and read the absorbance using an ELISA reader.

[0033] The binding efficacy of the above 15 rabbit anti-feline IgG monoclonal antibodies to the FPV whole-feline monoclonal antibody is as follows: Figure 2 As shown, the dose-response curve and regression model were successfully fitted, and all 15 expressed monoclonal antibodies could effectively bind to the whole cat-derived antibody. The five monoclonal antibodies with the best affinity, mAb-15, mAb-38, mAb-64, mAb-72 and mAb-94, were selected for subsequent validation.

[0034] 6. Indirect immunofluorescence detection of rabbit anti-cat IgG monoclonal antibody CRFK cells were revived in 24-well plates using CRFK growth medium. When the adherent CRFK cell coverage reached 70%–80%, the CRFK growth medium was replaced with FPV virus culture medium (the control group used fresh CRFK cell maintenance medium). The cells were then infected in a cell culture incubator at 37°C and 5% CO2 for 2 h. After infection, the culture medium was discarded, and the cells were replaced with CRFK cell maintenance medium and cultured in a cell culture incubator at 37°C and 5% CO2 for 2 days. After culture, the cells were washed twice with PBS, and then 500 μL of 4% paraformaldehyde was added to each well. The cells were incubated at room temperature for 30 min to fix their morphology. After fixation, the liquid portion was discarded, and the cells were washed three times with PBS. 500 μL of 3% Triton X-100 was added, and the cells were incubated at room temperature for 30 min before discarding the liquid. The cells were washed three times with PBS, and 1 mL of blocking buffer was added to each well. The cells were blocked at 37°C for 1 h, and then the blocking buffer was discarded. Add 500 μL of Myco-Protective Serum to each well, incubate at 37°C for 1 h, discard the liquid, and wash 5 times with PBS. Add 500 μL of FITC-rabbit anti-cat IgG (self-labeled) as secondary antibody to each well, incubate at 37°C for 1 h, discard the liquid, and wash 5 times with PBS. Add 500 μL of DAPI reagent to each well, react in the dark for 20 min, discard the liquid, wash 3 times with PBS, and finally add 30 μL of PBS to keep the cells moist. Observe the fluorescence signal of the cells using an inverted fluorescence microscope.

[0035] The results are as follows Figure 3 As shown, the binding ability of monoclonal antibodies mAb-15, mAb-38, mAb-64, mAb-72, and mAb-94 to feline IgG was verified using IFA (immunofluorescence assay). The results showed that all five monoclonal antibodies could specifically bind to feline IgG, with mAb-38 showing the most prominent green fluorescence signal. This indicates that mAb-38 is the most suitable for the specific detection of feline IgG.

[0036] The mAb-38 monoclonal antibody was sequenced, and its amino acid sequence is shown in Table 1.

[0037] Table 1. Amino acid sequence of mAb-38 monoclonal antibody

[0038] Example 2: Preparation of colloidal gold test strip for feline GnRH antibody detection 1. Preparation of colloidal gold solution Dissolve 1 g of chloroauric acid in ultrapure water and dilute to 50 mL. Filter to obtain a 2% chloroauric acid solution, which should be stored at 2–8 °C protected from light. Dissolve 1 g of trisodium citrate in ultrapure water and dilute to 50 mL. Filter to obtain a 2% trisodium citrate solution, which should be stored at 2–8 °C. Measure 98 mL of ultrapure water and 2 mL of the above 2% chloroauric acid solution, mix well, heat to boiling, and then quickly add 3.6 mL of the above 2% trisodium citrate solution. The color changes from colorless to black to wine red. After the color stabilizes, continue heating for 5 min, then stop heating. After the solution cools to room temperature, dilute to 100 mL to obtain a 0.04% colloidal gold solution.

[0039] The 0.04% colloidal gold solution was analyzed using a UV spectrophotometer. The solution was wine-red, transparent, and had no floating gold particles on the surface; the maximum absorption peak wavelength was approximately 521 nm. The 0.04% colloidal gold solution was stored at 2–8°C for no more than 6 months.

[0040] 2. Gold labeling process for rabbit anti-cat IgG Blocking solution: Weigh 2.5g of BSA, dissolve it thoroughly in ultrapure water, and bring the volume to 50 mL. Filter the solution and store it at 2~8℃ to obtain the blocking solution.

[0041] Reconstituted solution: Weigh 2g sucrose, 2g trehalose and 0.5g BSA, dissolve in 100mL of 0.01M Tris-HCl (pH 8.0), filter and store at 2~8℃ to obtain the reconstituted solution.

[0042] Take 1 mL of the above 0.04% colloidal gold solution, add 3 μL of 0.2 M K2CO3 solution to adjust the pH to 8, and mix for 15 min; add 28 μg of rabbit anti-cat IgG antibody mAb-38 prepared in Example 1, react at room temperature for 30 min, add 200 μL of blocking buffer and block at room temperature for 30 min; after blocking, centrifuge at 10000 ×g and 4℃ for 15 min, remove unbound colloidal gold particles and supernatant in sequence to obtain colloidal gold labeled precipitate, reconstitute with 200 μL of reconstitution solution to obtain colloidal gold labeled rabbit anti-cat IgG antibody solution, that is, colloidal gold-rabbit anti-cat IgG antibody conjugate solution.

[0043] 3. Preparation of GnRH-C-BSA Couplings A cysteine ​​residue was linked to the C-terminus of the GnRH peptide to obtain the GnRH-C peptide, whose amino acid sequence is QHWSYGLRPGC (SEQ ID NO.7). The GnRH-C peptide was synthesized by Shanghai Qiangyao Biotechnology Co., Ltd., and is a colorless and transparent liquid.

[0044] Dissolve 10 mg of BSA in PBS to a final concentration of 2 mg / mL to obtain a BSA solution. Dissolve 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC) in DMSO to a concentration of 13.4 mg / mL to obtain an SMCC solution. Add the BSA solution to the SMCC solution at a volume ratio of 1:40, react at room temperature for 15 min, and then replace the solvent with PBS using an ultrafiltration tube to obtain an SMCC-BSA solution.

[0045] GnRH-C and SMCC-BSA were mixed at a molar ratio of 10:1 and reacted at 4°C for 20-24 h. The GnRH-C-BSA conjugate was obtained by ultrafiltration and stored at -20°C.

[0046] 4. Preparation of sample pad Add appropriate amounts of Tween-20 and BSA to a 10 mM Tris-HCl (pH 8.0) solution to achieve final concentrations of 1% and 0.5%, respectively, to obtain the sample pad treatment solution. Lay a whole sheet of 8964 glass fiber flat on an acrylic plate, soak it in the above sample pad treatment solution for 30 min, and then dry it in a 45℃ drying oven for 24 h to obtain the sample pad.

[0047] 5. Preparation of the binding pad Weigh 2 g sucrose, 2 g trehalose, 0.5 g Tween-20, and 0.5 g sodium caseinate, dissolve them in 10 mM Tris-HCl (pH 8.0), and bring the volume to 100 mL to obtain the conjugation pad treatment solution. Pour the conjugation pad treatment solution into a tray containing RB65 glass fiber, soak for 15 min, remove the glass fiber, and dry it in a 45℃ oven for 4 h to obtain a blank conjugation pad, which should be sealed and stored away from light.

[0048] The above colloidal gold-rabbit anti-cat IgG antibody conjugate solution was sprayed onto the above blank conjugate pad at a rate of 6 μL / cm using a three-dimensional gold spraying spectrometer, and then dried in a 45°C drying oven for 5 h to obtain the conjugate pad.

[0049] 6. Preparation of nitrocellulose membranes 1 g of sucrose was dissolved in 100 mL of PBS solution to obtain a coating diluent. The prepared GnRH-C-BSA conjugate was diluted to 5 mg / mL using the coating diluent to prepare the test line solution, and streptococcal protein G was diluted to 0.5 mg / mL to prepare the control line solution. Using a three-dimensional gold sputtering spectrometer, the test line solution and control line solution were streaked onto a nitrocellulose membrane at 1 μL / cm intervals of 5 mm. The membrane was then dried at 45 °C for 5 h to obtain a nitrocellulose membrane with test and control lines.

[0050] 7. Assembly of test strips like Figure 4 The test strip is packaged as shown. The dried sample pad, conjugate pad, nitrocellulose membrane (NC membrane), and absorbent paper are sequentially overlapped and pasted onto a PVC base plate, then cut into strips and placed inside a plastic casing. One test strip and one packet of desiccant are sealed in an aluminum foil bag. The prepared test strip is used to detect GnRH positive and negative serum. 100 µL of sample is added to the sample well of the test strip, and the result is read after 10 minutes. Results are invalid after 20 minutes. The test results are as follows. Figure 5 As shown, this demonstrates the usability of the test strip.

[0051] Example 3: Optimization of Conditions for Cat GnRH Antibody Detection Colloidal Gold Test Strip 1. Investigation on the dosage of rabbit anti-cat IgG antibody mAb-38 The colloidal gold-rabbit anti-cat IgG antibody conjugate solution was prepared according to step 2 of Example 2, except that the amount of rabbit anti-cat IgG antibody mAb-38 added was adjusted to 12, 16, 22, and 28 μg sequentially to obtain different colloidal gold-rabbit anti-cat IgG antibody conjugate solutions. The colloidal gold test strip was assembled under otherwise unchanged conditions, and then 100 μL of feline GnRH-positive serum was loaded. After color development for 10 min, the results were as follows. Figure 6 As shown. Figure 6 As shown, within the dosage range of 12~28μg, the clarity of the detection line increased with the increase of the dosage of rabbit anti-cat IgG antibody mAb-38, and the detection line was most clear when the dosage of rabbit anti-cat IgG antibody mAb-38 was 28μg.

[0052] 2. Investigation on the coating concentration of GnRH-C-BSA conjugate on nitrocellulose membranes Nitrocellulose membranes were prepared according to step 6 of Example 2, except that the amount of GnRH-C-BSA was adjusted sequentially to prepare detection strip solutions with GnRH-C-BSA conjugate concentrations of 0.25, 0.5, 1, 2, and 5 mg / mL. The colloidal gold test strip was assembled under otherwise unchanged conditions, and then 100 μL of cat GnRH-positive serum was loaded. After 10 min of color development, the results were as follows: Figure 7 As shown. Figure 7 As shown, within the concentration range of 0.25–5 mg / mL, the sensitivity of the detection line increases with increasing solution concentration, reaching its highest sensitivity at a solution concentration of 5 mg / mL. Good detection results are also observed at a solution concentration of 2 mg / mL.

[0053] Example 4: Performance evaluation of the colloidal gold test strip for feline GnRH antibody detection 1. Specificity study The test strips prepared in Example 2 were used to detect feline GnRH-positive serum and feline panleukopenia virus (FPV)-positive serum, respectively. Each group was repeated three times to observe the specificity of the colloidal gold test strip for feline GnRH antibody detection. The test results are as follows: Figure 8 As shown, the third from the left represents the test results for feline GnRH-positive serum, and the third from the right represents the test results for feline panleukopenia virus (FPV)-positive serum. Figure 8 As can be seen, only the detection results of feline GnRH positive serum show two clear red bands, while the detection results of FPV positive serum show only one clear red band on line C, indicating that the feline GnRH antibody detection colloidal gold test strip of the present invention has good specificity.

[0054] 2. Sensitivity test Take 45 μL of feline GnRH-positive serum and add it to 1 mL of sample diluent (dilution ratio approximately 1:23), and continue to serially dilute in a 3-fold gradient to 1:439587. Use the test strip prepared in Example 2 for detection; the results are as follows. Figure 9 As shown. Among them, Figure 9 Result 1 is the test result at a dilution ratio of 1:23, Result 2 is the test result at a dilution ratio of 1:69, Result 3 is the test result at a dilution ratio of 1:201, Result 4 is the test result at a dilution ratio of 1:603, Result 5 is the test result at a dilution ratio of 1:1809, Result 6 is the test result at a dilution ratio of 1:5427, Result 7 is the test result at a dilution ratio of 1:16281, Result 8 is the test result at a dilution ratio of 1:48843, Result 9 is the test result at a dilution ratio of 1:146529, and Result 10 is the test result at a dilution ratio of 1:439587.

[0055] Depend on Figure 9 It can be seen that the colorimetric effect is best when the positive serum is diluted to 1:201 (i.e., 5 μL of serum is added to 1 mL of sample diluent). Figure 9 Result 3); When the positive serum was diluted to 1:146529, a faint red band was visible on the test line ( Figure 9 Result 9); When the positive serum was diluted to 1:439587, only a clear red band was visible at the control line ( Figure 9 (Result 10). The above results indicate that the feline GnRH antibody detection colloidal gold test strip of the present invention can detect feline GnRH positive serum diluted 1:146529, exhibiting strong sensitivity. Furthermore, the optimal colorimetric operation is to add 5 μL of serum to 1 mL of sample diluent.

[0056] 3. Stability test Intra-batch stability: Take the colloidal gold test strip for feline GnRH antibody detection prepared in the same batch, test feline GnRH positive serum, perform 3 replicates, and interpret the results.

[0057] Batch-to-batch stability: Three different batches of feline GnRH antibody detection colloidal gold test strips were used to detect GnRH-positive serum and the results were interpreted.

[0058] Test results as follows Figure 10 As shown, results 1-3 represent intra-batch stability test results, and results AC represent inter-batch stability test results. Intra-batch stability test results showed that the three repeated tests on feline GnRH positive serum yielded consistent results. Inter-batch stability test results showed that the results obtained by testing GnRH positive serum with test strips prepared from three different batches were consistent. These results indicate that the feline GnRH antibody detection colloidal gold test strip of the present invention exhibits good intra-batch and inter-batch stability.

[0059] Comparative Example 1 The colloidal gold test strip was prepared according to the following method: 1. Preparation of colloidal gold solution: Same as step 1 in Example 2.

[0060] 2. Preparation of GnRH-C-BSA conjugate: Same as step 3 in Example 2.

[0061] 3. Gold Standardization Process of GnRH-C-BSA Blocking solution: Weigh 2.5g of BSA, dissolve it thoroughly in ultrapure water, and bring the volume to 50 mL. Filter the solution and store it at 2~8℃ to obtain the blocking solution.

[0062] Reconstituted solution: Weigh 2g sucrose, 2g trehalose and 0.5g BSA, dissolve in 100mL of 0.01M Tris-HCl (pH 8.0), filter and store at 2~8℃ to obtain the reconstituted solution.

[0063] Take 1 mL of the above 0.04% colloidal gold solution, add 3 μL of 0.2 M K2CO3 solution to adjust the pH to 8, and mix for 15 min; add 28 μg of the above GnRH-C-BSA conjugate, react at room temperature for 30 min, add 200 μL of blocking solution and block at room temperature for 30 min; after blocking, centrifuge at 10000 ×g and 4℃ for 15 min, remove unbound colloidal gold particles and supernatant in sequence to obtain colloidal gold labeled precipitate, redissolve with 200 μL of redissolving solution to obtain colloidal gold labeled GnRH-C-BSA solution.

[0064] 4. Preparation of sample pad Add appropriate amounts of Tween-20 and BSA to a 10 mM Tris-HCl (pH 8.0) solution to achieve final concentrations of 1% and 0.5%, respectively, to obtain the sample pad treatment solution. Lay a whole sheet of 8964 glass fiber flat on an acrylic plate, soak it in the above sample pad treatment solution for 30 min, and then dry it in a 45℃ drying oven for 24 h to obtain the sample pad.

[0065] 5. Preparation of the binding pad Weigh 2 g sucrose, 2 g trehalose, 0.5 g Tween-20, and 0.5 g sodium caseinate, dissolve them in 10 mM Tris-HCl (pH 8.0), and bring the volume to 100 mL to obtain the conjugation pad treatment solution. Pour the conjugation pad treatment solution into a tray containing RB65 glass fiber, soak for 15 min, remove the glass fiber, and dry it in a 45℃ oven for 4 h to obtain a blank conjugation pad, which should be sealed and stored away from light.

[0066] The colloidal gold-labeled GnRH-C-BSA solution was sprayed onto the blank conjugate pads at a rate of 6 μL / cm using a three-dimensional gold spraying spectrometer, and then dried in a 45°C drying oven for 5 h to obtain the conjugate pads.

[0067] 6. Preparation of nitrocellulose membranes 1 g of sucrose was dissolved in 100 mL of PBS solution to obtain a coating diluent. The rabbit anti-cat IgG antibody mAb-38 prepared in Example 1 was diluted with the coating diluent to concentrations of 3, 6, and 9 mg / mL as test line solutions, respectively. Streptococcal protein G was diluted to 0.5 mg / mL as a control line solution. Using a three-dimensional gold sputtering spectrometer, the above test line solutions and control line solutions were streaked onto a nitrocellulose membrane at 1 μL / cm intervals of 5 mm. The membrane was then dried at 45°C for 5 h to obtain a nitrocellulose membrane with test lines and control lines.

[0068] 7. Assembly of test strips: Following step 7 of Example 2, three colloidal gold test strips were assembled. The prepared test strips were used to detect feline GnRH-positive and negative sera, respectively. The test results are as follows: Figure 11 As shown, positive serum did not develop color, so the test strip is unusable.

[0069] Comparative Example 2 The colloidal gold test strip was prepared according to the following method: 1. Preparation of colloidal gold solution: Same as step 1 in Example 2.

[0070] 2. Gold labeling process for rabbit anti-cat IgG: Same as step 2 in Example 2.

[0071] 3. Preparation of C-GnRH-BSA conjugates A cysteine ​​residue was linked to the N-terminus of the GnRH peptide to obtain the C-GnRH peptide, whose amino acid sequence is CQHWSYGLRPG (SEQ ID NO. 8). The C-GnRH peptide was synthesized by Shanghai Qiangyao Biotechnology Co., Ltd., and is a colorless and transparent liquid.

[0072] Dissolve 10 mg of BSA in PBS to a final concentration of 2 mg / mL to obtain a BSA solution. Dissolve 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC) in DMSO to a concentration of 13.4 mg / mL to obtain an SMCC solution. Add the BSA solution to the SMCC solution at a volume ratio of 1:40, react at room temperature for 15 min, and then replace the solvent with PBS using an ultrafiltration tube to obtain an SMCC-BSA solution.

[0073] C-GnRH and SMCC-BSA were mixed at a molar ratio of 10:1 and reacted at 4℃ for 20-24 h. The C-GnRH-BSA conjugate was obtained by ultrafiltration and stored at -20℃.

[0074] 4. Preparation of sample pad: Same as step 4 in Example 2.

[0075] 5. Preparation of the binding pad: Same as step 5 in Example 2.

[0076] 6. Preparation of nitrocellulose membranes 1 g of sucrose was dissolved in 100 mL of PBS solution to obtain a coating diluent. The prepared C-GnRH-BSA conjugate was diluted with the coating diluent to prepare test line solutions of 2.5, 5, and 7.66 mg / mL, respectively. Streptococcal protein G was diluted to 0.5 mg / mL as a control line solution. Using a three-dimensional gold sputtering spectrometer, the test line solutions and control line solutions were streaked onto a nitrocellulose membrane at 1 μL / cm intervals of 5 mm. The membranes were then dried at 45°C for 5 h to obtain nitrocellulose membranes with test lines and control lines.

[0077] 7. Assembly of test strips: Following step 7 of Example 2, three colloidal gold test strips were assembled. The prepared test strips were used to detect feline GnRH-positive and negative sera, respectively. The test results are as follows: Figure 12 As shown, the colorimetric effect was poor when the concentration of the test strip solution was 2.5 mg / mL; and false positives occurred with negative serum at test strip solution concentrations of 5 mg / mL and 7.66 mg / mL, rendering the test strip unusable.

[0078] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A colloidal gold test strip for detecting feline GnRH antibodies, characterized in that, The colloidal gold test strip includes a PVC base plate, a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper; the sample pad, conjugate pad, nitrocellulose membrane, and absorbent paper are sequentially adhered to the PVC base plate and connected by overlapping; detection lines and control lines are sequentially distributed on the nitrocellulose membrane along the direction from the conjugate pad to the absorbent paper; The conjugate pad is coated with a colloidal gold-rabbit anti-cat IgG antibody conjugate solution; The detection line is adsorbed with a detection line solution, which includes a GnRH-C-BSA conjugate. The GnRH-C-BSA conjugate is a conjugate of a GnRH polypeptide with a cysteine ​​residue linked to its C-terminus and bovine serum albumin (BSA). The control line is adsorbed with control line solution, which includes streptococcal protein G.

2. The colloidal gold test strip according to claim 1, characterized in that, The amino acid sequence of VH-CDR1 of the rabbit anti-cat IgG antibody is shown in SEQ ID NO.1, the amino acid sequence of VH-CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of VH-CDR3 is shown in SEQ ID NO.

3. The amino acid sequence of VL-CDR1 of the rabbit anti-cat IgG antibody is shown in SEQ ID NO.4, the amino acid sequence of VL-CDR2 is shown in SEQ ID NO.5, and the amino acid sequence of VL-CDR3 is shown in SEQ ID NO.

6.

3. The colloidal gold test strip according to claim 1 or 2, characterized in that, In the colloidal gold-rabbit anti-cat IgG antibody conjugate, the weight ratio of the colloidal gold to the rabbit anti-cat IgG antibody is 400:(12~28). And / or, in terms of the concentration of colloidal gold, the concentration of the colloidal gold-rabbit anti-cat IgG antibody conjugate solution is 1~5 mg / mL; And / or, the amount of the colloidal gold-rabbit anti-cat IgG antibody conjugate solution sprayed onto the binding pad is 2~7 μL / cm.

4. The colloidal gold test strip according to claim 1, characterized in that, In the GnRH-C-BSA conjugate, the amino acid sequence of the GnRH-C polypeptide is shown in SEQ ID NO.7; And / or, the molar ratio of the GnRH-C polypeptide to BSA is (8~12):1; And / or, the concentration of the GnRH-C-BSA conjugate in the detection line solution is 0.25~5 mg / mL; And / or, the amount of the detection line solution on the detection line is 0.5~1.5 μL / cm.

5. The colloidal gold test strip according to claim 1, characterized in that, The concentration of streptococcal protein G in the control solution is 0.25~1.5 mg / mL; And / or, the amount of the control line solution used for streaking on the control line is 0.5~1.5 μL / cm.

6. The colloidal gold test strip according to claim 1, characterized in that, Along the long side of the colloidal gold test strip, the length of the sample pad is 10-15 mm, the length of the conjugation pad is 10-15 mm, the length of the nitrocellulose membrane is 20-30 mm, and the length of the absorbent paper is 12-20 mm. On the nitrocellulose membrane, the distance between the detection line and the conjugation pad is 10-15 mm; the distance between the detection line and the control line is 4-8 mm; and the distance between the control line and the absorbent paper is 4-8 mm.

7. The colloidal gold test strip according to claim 1, characterized in that, The preparation process of the colloidal gold-rabbit anti-cat IgG antibody conjugate includes: Adjust the pH of the colloidal gold solution to 4-10 and mix well for 5-30 minutes. Add rabbit anti-cat IgG antibody and react for 30-60 minutes. Then add blocking solution and block for 30-60 minutes. Separate the solid and liquid and take the solid.

8. The colloidal gold test strip according to claim 1, characterized in that, The preparation process of the GnRH-C-BSA conjugate includes: SMCC is reacted with BSA to obtain SMCC-BSA; The GnRH-C peptide is reacted with the SMCC-BSA to obtain the GnRH-C-BSA conjugate.

9. A method for preparing the colloidal gold test strip according to any one of claims 1 to 8, comprising the following steps: (1) A colloidal gold-rabbit anti-cat IgG antibody conjugate solution was sprayed onto a blank conjugate pad to obtain a conjugate pad; (2) Draw lines on the nitrocellulose membrane in sequence to adsorb the detection line solution and the quality control line solution, and obtain the nitrocellulose membrane after the lines are drawn; (3) The sample pad, conjugate pad, crisscrossed nitrocellulose membrane and absorbent paper are then pasted onto the PVC base plate in an overlapping manner.

10. A kit for detecting feline GnRH antibodies, characterized in that, The kit comprises the colloidal gold test strip as described in any one of claims 1 to 8.

Images