WO2017075937A1 - 无乳链球菌的胶体金快速检测试纸 - Google Patents

无乳链球菌的胶体金快速检测试纸 Download PDF

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WO2017075937A1
WO2017075937A1 PCT/CN2016/078451 CN2016078451W WO2017075937A1 WO 2017075937 A1 WO2017075937 A1 WO 2017075937A1 CN 2016078451 W CN2016078451 W CN 2016078451W WO 2017075937 A1 WO2017075937 A1 WO 2017075937A1
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antibody
pad
cctcc
colloidal gold
streptococcus agalactiae
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PCT/CN2016/078451
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English (en)
French (fr)
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肖丹
刘天强
黄冠军
阳涛
刘衍鹏
戴景辉
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通威股份有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56944—Streptococcus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody

Definitions

  • the invention relates to a colloidal gold rapid test strip for Streptococcus agalactiae.
  • Tilapia is a tropical fish native to Africa. It was introduced from abroad in the world in the 1970s and 1980s, and breeds with rapid growth, high yield, and strong disease resistance were selected. At present, China is not only the largest producer of tilapia in the world, but also the largest exporter of tilapia. Domestic tilapia culture is concentrated in southern areas such as Guangdong, Hainan, Guangxi and Fujian. In 2009, tilapia “extrusion syndrome” began to occur in the tilapia aquaculture area. The disease is fierce, the incidence rate is as high as 35%, the mortality rate of the diseased fish is close to 100%, and the disease lasts for a long time. The condition, the farmers suffered a lot of pain.
  • the invention provides a colloidal gold rapid test strip for Streptococcus agalactiae.
  • Colloidal gold immunochromatographic test strip using nitrocellulose membrane as solid phase carrier, using colloidal gold as a tracer marker, binding to antibody, and utilizing antigen-antibody reaction under diafiltration or capillary action of microporous membrane
  • the high specificity and colloidal gold-specific color traces the binding of the gold standard antibody to the antigen or secondary antibody, showing red bands or spots visible to the naked eye, enabling qualitative or quantitative analysis of the analyte.
  • the double antibody colloidal gold immunochromatographic test paper of the invention comprises: using a nitrocellulose membrane as a solid phase carrier, using colloidal gold as a tracer label, and the specific antibody 1 is fixed in a strip shape on the nitrocellulose membrane, and A specific antibody 2 is combined with a colloidal gold solution to form a gold-labeled antibody.
  • the sample to be tested When the sample to be tested is added to the sample pad at one end of the test strip, it moves forward by capillary action, dissolves the gold-labeled antibody on the binding pad, reacts with each other, and then moves to the detection zone where the antibody 1 is immobilized, the analyte and the gold Complex antibody formation
  • the compound is specifically bound to it and trapped, gathered on the test strip, and a red band visible to the naked eye is obtained through a visually detectable colloidal gold marker, thereby realizing qualitative or quantitative analysis of the test object.
  • the colloidal gold immunochromatographic test strip of Streptococcus agalactiae is a colloidal gold immunochromatographic test paper developed based on the principle of double antibody sandwich detection, wherein the two antibodies used are the deposit numbers preserved by the Chinese typical culture center: CCTCC NO: The antibody secreted by the cell line of C2015177 and the accession number deposited by the China National Culture Center: CTCCC NO: antibody secreted by the cell line of C2015178.
  • CCTCC NO: C2015177 was deposited at the China Center for Cultivated Culture (CCTCC) on October 21, 2015, and its address is: Wuhan University, Wuhan, China.
  • CCTCC NO: C2015178 was deposited at the China Center for Cultivated Culture (CCTCC) on October 21, 2015, and its address is: Wuhan University, Wuhan, China.
  • the antibody deposited by the Chinese classical culture center deposit: CCTCC NO: C2015177 is used to prepare the gold-labeled antibody; the Chinese classical culture center deposit number is secreted by the cell line of CCTCC NO: C2015178.
  • the antibody is used to coat a nitrocellulose membrane to form a detection zone.
  • the preparation method of the antibody is as follows:
  • the purification is purified using a Protein G Sepharose affinity chromatography column.
  • the test paper has the following structure: comprising a PVC bottom plate and a nitrocellulose membrane disposed thereon, the sample pad is connected to one end of the nitrocellulose membrane through a colloidal gold pad, and the other end of the nitrocellulose membrane is overlapped with an absorbent pad.
  • a detection zone is disposed on a side of the nitrocellulose membrane adjacent to the colloidal gold pad, and a quality control zone is disposed on a side adjacent to the absorbent pad.
  • the sample pad is overlapped on the upper surface of one end of the colloidal gold pad, and the other end of the colloidal gold pad is overlapped on the upper surface of the nitrocellulose membrane.
  • control region is coated with a layer of goat anti-mouse polyclonal antibody.
  • the invention also provides a method for preparing the aforementioned test strip, the steps are as follows:
  • the invention also provides an immunoassay kit for detecting Streptococcus agalactiae, which comprises the following two monoclonal antibodies against Streptococcus agalactiae: the two S. agalactiae antibodies used are the deposit numbers preserved by the Chinese typical culture center. : CCTCC NO: The antibody secreted by the cell line of C2015177 and the deposited sample of the Chinese Culture Center: the antibody secreted by the cell line of CCTCC NO: C2015178.
  • the preparation method of the antibody is as follows:
  • step (2) the purification is carried out using a Protein G Sepharose affinity chromatography column.
  • the kit further includes an immunofluorescence detecting reagent, a radioimmunoassay reagent, an enzyme-linked immunosorbent assay reagent, or an immunogold colloid detecting reagent.
  • the two monoclonal antibodies of the invention can be effectively combined with Streptococcus agalactiae, and the coloring substances with the coloring are obvious, and can be combined with any conventional reagents for immunoassay techniques to detect Streptococcus agalactiae.
  • the existing immunoassay technologies include immunofluorescence technology, radioimmunoassay technology, enzyme-linked immunosorbent assay technology and immunogold colloid technology.
  • the two monoclonal antibodies of the present invention can be respectively combined with conventional immunofluorescence detection reagents, radioimmunoassay reagents, enzymes.
  • the combination of immunosorbent assay reagent or immunogold colloid detection reagent uses immunofluorescence technique, radioimmunoassay technique, enzyme-linked immunosorbent assay or immunogold colloid technology to accurately detect Streptococcus agalactiae.
  • Immunofluorescence technique An immunofluorescence technique is a method of detecting a corresponding antigen (or antibody) in a tissue, a cell or a serum using a fluorescein-labeled antibody (or antigen).
  • Radioimmunoassay technology Radioimmunoassay technology is currently the most sensitive detection technology. It uses radioactive allo-labeled antigen (or antibody) to bind to the corresponding antibody (or antigen) and then determine the radioactive detection result of antigen-antibody conjugate. .
  • Enzyme-linked immunosorbent assay ELISA
  • Enzyme-linked immunosorbent assay Enzyme-linked immunosorbent assay (ELISA) is currently the most widely used immunoassay.
  • the method is to label the secondary antibody with an enzyme, and the specificity of the antigen-antibody reaction is combined with the action of the enzyme-catalyzed substrate, and the color change of the color after the substrate is applied according to the enzyme. To judge the test results, the sensitivity can reach ng level.
  • Commonly used enzymes for labeling are horseradish peroxidase (HRP), alkaline phosphatase (AP), and the like.
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • Immunogold colloid technology This method is to label the secondary antibody with colloidal gold particles, and utilize the specific reaction between the antigen and antibody to finally adsorb the colloidal gold-labeled secondary antibody on the percolation membrane. This method is simple, rapid and extensive. Used in clinical screening.
  • the invention also provides the use of the aforementioned test strips and test kits for the preparation of detecting Streptococcus agalactiae.
  • the invention also provides a method for detecting Streptococcus agalactiae, the steps are as follows: using the sample pad of the test strip of the invention to infiltrate the sample to be inspected, and observing whether the detection area and the quality control area are colored.
  • the invention also provides a method for detecting Streptococcus agalactiae, the steps are as follows: taking the sample to be tested, and detecting by using the test kit described above.
  • the sample to be inspected is a fish body or a culture water body.
  • the colloidal gold rapid test strip prepared by the monoclonal antibody of Streptococcus agalactiae has the advantages of high sensitivity, good specificity, convenient and rapid operation, and solves the problem of the detection of the existing Streptococcus agalactiae, and has a good application prospect.
  • Figure 2 structure of the test paper 1: PVC substrate; 2: nitrocellulose membrane; 3: colloidal gold pad; 4: absorbent pad; 5: detection zone; 6: quality control zone; 7: sample pad;
  • Figure 3 1: 6 ⁇ 10 10 CFU / ml; 2: 6 ⁇ 10 9 CFU / ml; 3: 6 ⁇ 10 8 CFU / ml; 4: 6 ⁇ 10 7 CFU / ml; 5: 6 ⁇ 10 6 CFU / M line; C Line: quality control line; T Line: test line;
  • Figure 4 shows the specificity test strip
  • Figure 5 shows the results of detection of diseased fish tissues and water bodies.
  • Streptococcus agalactiae TW3 is used for cloning and preparation of recombinant antigens, Streptococcus agalactiae (ATCC51487), Streptococcus agalactiae C918 (bovine source), Streptococcus agalactiae TW7 (fish source), Streptococcus agalactiae TW10 (fish) Source), E.
  • the amplified sip DNA was ligated into a pET32a vector containing a sequence encoding hexahistidine.
  • Escherichia coli BL21 was transformed with pET32a-sip.
  • Transformants were cultured in LB liquid medium containing 100 ⁇ g/mL kanamycin at 37 °C. 0.8 mM isopropyl- ⁇ -D-thiogalactoside (IPTG) was added and incubated for an additional 5 h at 37 ° C to express the hexahistidine-tagged recombinant Sip (rSip).
  • the soluble fraction containing rSip was extracted by ultrasonic shaking, then purified and eluted using an IMAC affinity chromatography column, and the protein concentration was determined using a bisquinolinecarboxylic acid detection kit (Sigma-Aldrich).
  • amino acid sequence of rSip (SEQ ID NO. 1) is as follows:
  • Emulsification with a complete adjuvant (Sigma-Aldrich) (emulsification method: mixing the antigen and Freund's complete adjuvant in equal volume, inhaling into a syringe, removing the syringe needle, connecting another syringe with a hose, and tying it 25 BA, 25 ⁇ g, 100 ⁇ g, and 150 ⁇ g rSip protein were intraperitoneally injected into female BALB/c mice with a fixed hose, which was used to push the two syringes in turn, allowing the mixture to flow back and forth between the syringes through the hose. 8 weeks old).
  • mice were re-injected (emulsification method and injection dose as before) with the same antigen emulsified by Freund's incomplete adjuvant (Sigma-Aldrich).
  • the rSip protein was injected intraperitoneally once (injection dose as before).
  • the spleen cells of the immunized mice were obtained (isolation method: high-free BALB/c mice were taken, and after the neck was sacrificed, immersed in 75% alcohol for 3 to 5 minutes, aseptic operation to open the abdominal cavity, and be careful Remove the spleen into the plate, rinse with a small amount of DMEM basal medium for 2 to 3 times, and carefully remove the connective tissue around the spleen. Then pour the serum-free plate into the other dish and cover the copper mesh on it, and take the spleen on the copper. On the net, gently grind with a grinding rod. After the cells are completely released, the spleen cells are collected and centrifuged at 1000 rpm for 10 min).
  • spleen cells and myeloma cells S/P20 were fused in a ratio of 5:1 with 50% (w/v) polyethylene glycol 4000 (Sigma-Aldrich) (step of cell fusion) :S/P20 cells were donated by Prof. Rao Chaolong from Chengdu University of Traditional Chinese Medicine. Fusion procedure: S/P20 cells in logarithmic growth phase were mixed well with spleen cells, centrifuged at 1000 rpm for 10 min, the supernatant was discarded, and the palms were gently centrifuged. Bottle bottom, break up the precipitated cells.
  • the obtained hybridoma cells were cultured in hypoxanthine-aminopterin-thymidine medium, and 5 ⁇ g/ml of rSip was used as a coating antigen, and the antibody in the culture supernatant was detected by ELISA to select hybridoma cells to obtain Hybridoma cells positive for ELISA were identified.
  • Hybridoma cells identified as positive by ELISA were propagated in ascites of BALB/c mice.
  • the ascites was collected and purified using a Protein G Sepharose affinity chromatography column (GE Healthcare Life Sciences) (purification method: the mouse ascites was aspirated by a syringe, and the larger clot was removed by centrifugation at 12000 g for 15 min at 4 ° C.
  • the ascites was diluted 10-fold with 0.02 M pH 7.0 in PBS buffer, filtered through a 0.45 ⁇ m filter, and then loaded onto a Protein G affinity column equilibrated with 0.02 M pH 7.0 in PBS buffer using 0.1 M pH 2.7.
  • Gly-HCl was eluted as the eluent, the elution peak was collected, and the pH was adjusted to about 7.0 with 1.0 M Tris-HCl, pH 9.0, and finally dialysis desalted and lyophilized) rSip mAb in ascites.
  • Another mAb was diluted to 2 mg/ml with 0.1 M PBS.
  • Sheep anti-mouse IgG was diluted to 1.0 mg/ml with 0.1 M PBS.
  • the former is used to coat the detection zone on the NC membrane and the latter is used in the quality control zone.
  • the membrane was held at 37 ° C for 24 h. After blocking with BB buffer (Artron BioResearch Inc), the membrane was washed with WB buffer and then dried at room temperature.
  • test strip is assembled with a sample pad, a bonding pad, a reaction film, and an adsorption pad.
  • the test paper was cut into 3 mm strips using an automatic slitter.
  • All bound-mAbs were paired with coated-mAbs for test strip preparation.
  • the sample pad of the test strip was inserted into the S. agalactiae sample until half of the NC membrane was immersed in the liquid. The formation of the quality control zone and the detection zone was observed after 3-10 min at room temperature. Paired antibodies showing the strongest positive signal for S. agalactiae and negative results for other tested bacteria were screened.
  • a total of 84 positive cell lines were obtained after cell fusion. All 84 monoclonal antibodies were purified and used as coated antibody/binding antibodies for test strip assembly. After multiple pairing tests, the NC7b-CE12 pairing was found to have the strongest signal for S. agalactiae and no signal for other bacteria.
  • hybridoma cell line TWDB-WR-1 and the hybridoma cell line TWDB-WR-2 were separately prepared as follows: monoclonal antibody NC7b and monoclonal antibody CE12 were prepared as follows:
  • the hybridoma cell strain was propagated in the ascites of BALB/c mice, specifically, sterilized liquid paraffin was intraperitoneally injected into 8 weeks old BALB/c mice, 0.5 ml/mouse. After 7 days, the above two hybridoma cell lines were suspended in serum-free RPMI-1640 medium, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the serum in the medium was washed away, and then the appropriate amount of serum-free RPMI-1640 medium was used.
  • suspending cell pellets were injected into the abdominal cavity of mice per 0.5ml (containing about 10 6 hybridoma cells collected ascites (apparent abdominal swelling taken 7 days after ascites in mice), using Protein G Sepharose affinity column (GE Healthcare Life Sciences) Purification (purification of the specific steps: the mouse ascites was aspirated by a syringe, and the larger clot was removed by centrifugation at 12000 g for 15 min at 4 ° C. The treated ascites was buffered with 0.02 M pH 7.0 PBS. The solution was diluted 10 times, filtered through a 0.45 ⁇ m filter, and then loaded onto a protein G affinity chromatography column equilibrated with 0.02 M pH 7.0 in PBS buffer.
  • reaction was carried out with 0.1 M Gly-HCl pH 2.7 as the eluent. Elution was performed, the elution peak was collected, and the pH was adjusted to about 7.0 with 1.0 M Tris-HCl, pH 9.0, and finally desalinated and lyophilized) rSip mAb in ascites.
  • test strip of the invention is a double antibody sandwich test strip, and the test paper structure diagram is shown in Figure 2:
  • colloidal gold is mainly prepared by the trisodium citrate reduction method. 150 ⁇ l of sodium citrate was added to a 10 mL system of chloroauric acid solution. The colloidal gold particles were visually observed to be uniform in color, showing a red wine color and a clear halo. The size of the colloidal gold particles was consistent by electron microscopy, and the particle diameter was about 20 nm. The UV-scanned gold particles have a narrow peak width, indicating that the colloidal gold solution has good dispersibility.
  • the labeled gold standard antibody is coated on the glass fiber membrane to form a colloidal gold pad: the gold standard antibody solution is added to the nozzle storage bottle of the spraying machine, the glass fiber membrane is sprayed, and dried, which is a colloidal gold pad.
  • Test paper assembly (structure shown in Figure 2): nitrocellulose membrane (2) attached to PVC substrate (1), sample pad (7) is connected to one end of nitrocellulose membrane (2) through colloidal gold pad (3).
  • the other end of the nitrocellulose membrane (2) is overlapped with an absorbent pad (4), and the side of the nitrocellulose membrane (2) adjacent to the colloidal gold pad (3) is coated with the monoclonal antibody NC7b of the present invention (Step 1 Preparation)
  • the detection zone (5) the side close to the absorbent pad (4) is coated with goat anti-mouse IgG as a quality control zone (6).
  • the method of coating the antibody on the colloidal gold pad taking the antibody, preparing the antibody solution, adding it to the nozzle storage bottle of the spraying machine, spraying the test paper, and drying.
  • the S. agalactiae was diluted to a ratio of 6 x 10 10 CFU/ml to 6 x 10 6 CFU/ml, and then tested with a test strip (i.e., the test strip established in Example 1 of the present invention) to evaluate the minimum detected amount.
  • a test strip i.e., the test strip established in Example 1 of the present invention
  • Test strips for testing Streptococcus agalactiae (ATCC 51487), Streptococcus agalactiae C918 (bovine source), E. faecalis, Enterococcus faecalis, Streptococcus faecalis, Aeromonas guinea, S.
  • the test method using the test paper of the invention using the sample pad of the test strip of the invention to infiltrate the sample to be inspected, and observing whether the detection area and the quality control area develop color.
  • the sample to be inspected is positive for detection; if only the red strip appears in the position of the quality control area 6, and there is no position in the detection area 5
  • the sample to be tested is negative for detection; if there is no red strip at the position of the quality control area 6 of the test strip, the detection result is invalid regardless of whether there is a red strip at the position of the detection area.
  • the S. agalactiae broth was diluted 10-fold to 6 ⁇ 10 10 CFU/ml to 6 ⁇ 10 6 CFU/ml to measure the sensitivity of the test strip.
  • concentration was 6 ⁇ 10 7 CFU/ml
  • a weak T line was observed, but when the concentration continued to fall to 6 ⁇ 10 6 CFU/ml, the T line did not appear (Fig. 3).
  • Figure 3 shows that the detection limit of the paired antibody is 6 x 10 7 CFU/ml.
  • the water samples of the affected tilapia and the diseased pond were tested using the S. agalactiac test.
  • the results showed that the diseased fish contained a large amount of Streptococcus agalactiae, but no Streptomyces agalactiae was detected in the water (Fig. 5). Consistent with the results of the PCR test (not provided), suggesting that Streptococcus agalactiae has proliferated in the fish, causing disease.
  • test results show that the S. agalactiae test strip of the invention has high sensitivity, high specificity and good repeatability, and can accurately detect actual samples.

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Abstract

一种无乳链球菌的双抗体夹心法胶体金快速检测试纸，其中采用的两种抗体为中国典型培养物中心保藏的保藏号：CCTCC NO：C2015177的细胞株分泌的抗体以及中国典型培养物中心保藏的保藏号：CCTCC NO：C2015178的细胞株分泌的抗体。上述无乳链球菌单克隆抗体制备的胶体金快速检测试纸灵敏度高，特异性好，操作方便、快速，解决了现有无乳链球菌检测的问题，应用前景良好。

Description

无乳链球菌的胶体金快速检测试纸 技术领域
本发明涉及一种无乳链球菌的胶体金快速检测试纸。
背景技术
罗非鱼是原产于非洲的热带鱼类，上世界七八十年代我国多次从国外引种，并选育出长势快、产量高、抗病力强的品种，此后迅速成功推广养殖。目前，我国不仅是世界上最大的罗非鱼养殖生产国，也是最大的罗非鱼出口国。国内罗非鱼养殖集中在广东、海南、广西、福建等南方地区。2009年开始在罗非鱼养殖密集区先后暴发罗非鱼“突眼症”，该病来势凶猛，发病率高达35％，发病鱼死亡率接近100％，且病害持续时间长，药物防治难以控制病情，养殖户损失惨痛。
及时准确的发现和诊断病原，是成功预防和治疗罗非鱼链球菌病的前提。目前，对于无乳链球菌的诊断，主要采用(1)分子生物学的方法，如PCR法进行诊断，该类方法依赖于贵重的仪器设备和专业的技术人员，检测时间长达4-6个小时，非常不利于在基层推广运用。(2)常规的分离培养法，依赖于专业操作人员的显微镜形态判断，容易造成误判，此类方法的培养时间长达18-24个小时，也不适用于无乳链球菌的快速检测。
发明内容
本发明提供了一种无乳链球菌的胶体金快速检测试纸。
胶体金免疫层析检测试纸：是以硝酸纤维素膜为固相载体，以胶体金作为示踪标记物，与抗体结合，在微孔膜的渗滤作用或毛细管作用下，利用抗原抗体的反应的高度特异性和胶体金特有的颜色对金标抗体与抗原或二抗的结合进行示踪，显示肉眼可见的红色条带或斑点，从而实现对待测物的定性或定量分析。
本发明双抗体胶体金免疫层析试纸：即是以硝酸纤维素膜为固相载体，以胶体金作为示踪标记物，特异性的抗体1以条带状固定在硝酸纤维素膜上，另一特异性的抗体2与胶体金溶液结合形成的金标抗体。当待测样品加到试纸条一端的样品垫上后，通过毛细作用向前移动，溶解结合垫上的金标抗体，相互反应后再移动至固定有抗体1的检测区时，待测物和金标抗体形成的复 合物又与之发生特异性结合而被截留，聚集在检测带上，通过可目测的胶体金标记物得到肉眼可见的红色条带，从而实现对待测物的定性或定量分析。
本发明无乳链球菌的胶体金免疫层析检测试纸，它是基于双抗体夹心检测原理研制的胶体金免疫层析试纸，其中，采用的两种抗体为中国典型培养物中心保藏的保藏号：CCTCC NO：C2015177的细胞株分泌的抗体以及中国典型培养物中心保藏的保藏号：CCTCC NO：C2015178的细胞株分泌的抗体。
本发明保藏号为：CCTCC NO：C2015177的细胞株，于2015年10月21日保藏在中国典型培养物中心(CCTCC)，其地址为：中国.武汉.武汉大学。
本发明保藏号为：CCTCC NO：C2015178的细胞株，于2015年10月21日保藏在中国典型培养物中心(CCTCC)，其地址为：中国.武汉.武汉大学。
其中，所述中国典型培养物中心保藏的保藏号：CCTCC NO：C2015177的细胞株分泌的抗体用于制备金标抗体；中国典型培养物中心保藏的保藏号：CCTCC NO：C2015178的细胞株分泌的抗体用于包被硝酸纤维素膜，形成检测区。
优选地，所述抗体的制备方法如下：
(1)分别取保藏号：CCTCC NO：C2015177的细胞株和保藏号：CCTCC NO：C2015178的杂交瘤细胞株，注射入BALB/c小鼠腹水中增殖；
(2)收集腹水，纯化，即可。
优选地，步骤(2)中，所述纯化是是采用Protein G Sepharose亲和层析柱纯化。
优选地，所述试纸的结构如下：包括PVC底板和设于其上的硝酸纤维素膜，样品垫通过胶体金垫与硝酸纤维素膜一端连接，硝酸纤维素膜另一端搭接有吸水垫，所述硝酸纤维素膜上靠近胶体金垫的一侧设有检测区，靠近吸水垫的一侧设有质控区。
优选地，所述样品垫搭接在胶体金垫一端上表面，胶体金垫另一端搭接在硝酸纤维素膜上表面。
进一步优选地，所述质控区上包被有一层羊抗鼠的多克隆抗体层。
本发明还提供了制备前述检测试纸的方法，步骤如下：
a、取中国典型培养物中心保藏的保藏号：CCTCC NO：C2015177的细 胞株分泌的抗体，与胶体金混合，制备金标抗体，喷涂于玻璃纤维膜上，制得胶体金垫；
b、取硝酸纤维素膜，粘贴于PVC底板(1)上，将样品垫通过胶体金垫与硝酸纤维素膜的一端连接，再在硝酸纤维素膜的另一端搭接吸水垫；
c、再在硝酸纤维素膜靠近胶体金垫的一侧喷涂取中国典型培养物中心保藏的保藏号：CCTCC NO：C2015178的细胞株包被的抗体作为检测区，在靠近吸水垫的一侧包被羊抗鼠IgG作为质控区。
本发明还提供了一种检测无乳链球菌的免疫检测试剂盒，它包括如下两种无乳链球菌单克隆抗体：采用的两种无乳链球菌抗体为中国典型培养物中心保藏的保藏号：CCTCC NO：C2015177的细胞株分泌的抗体以及中国典型培养物中心保藏的保藏号：CCTCC NO：C2015178的细胞株分泌的抗体。
所述抗体的制备方法如下：
(1)分别取保藏号：CCTCC NO：C2015177的细胞株和保藏号：CCTCC NO：C2015178的杂交瘤细胞株，注射入BALB/c小鼠腹水中增殖；
(2)收集腹水，纯化，即可。
步骤(2)中，所述纯化是采用Protein G Sepharose亲和层析柱纯化。
优选地，所述试剂盒还包括免疫荧光检测用试剂、放射免疫检测用试剂、酶联免疫吸附检测用试剂或免疫金胶体检测用试剂。
本发明两种单克隆抗体可以与无乳链球菌有效结合，带有的显色物质显色明显，可以与现有任意的免疫检测技术常规用试剂联用，检测无乳链球菌。
现有的免疫检测技术包括免疫荧光技术、放射免疫检测技术、酶联免疫吸附技术和免疫金胶体技术，本发明两种单克隆抗体可以分别与常规的免疫荧光检测试剂、放射免疫检测试剂、酶联免疫吸附检测试剂或免疫金胶体检测试剂联合使用，使用免疫荧光技术、放射免疫检测技术、酶联免疫吸附技术或免疫金胶体技术，准确检测无乳链球菌。
1.免疫荧光技术：免疫荧光技术是利用荧光素标记的抗体(或抗原)检测组织、细胞或血清中的相应抗原(或抗体)的方法。2.放射免疫检测技术：放射免疫检测技术是目前灵敏度最高的检测技术，利用放射性同素标记抗原(或抗体)，与相应抗体(或抗原)结合后，通过测定抗原抗体结合物的放射检测结果。3.酶联免疫吸附检测技术(ELISA)：酶联免疫检测是目前应用最广泛的免疫检测方法。该方法是将二抗标记上酶，抗原抗体反应的特异性与酶催化底物的作用结合起来，根据酶作用底物后的显色颜色变化 来判断试验结果，其敏感度可达ng水平。常见用于标记的酶有辣根过氧化物酶(HRP)、碱性磷酸酶(AP)等。4.免疫金胶体技术：该方法是将二抗标记上胶体金颗粒，利用抗原抗体间的特异性反应，最终将胶体金标记的二抗吸附于渗滤膜上，此方法简单，快速，广泛应用于临床筛查。
本发明还提供了前述的检测试纸和检测试剂盒在制备检测无乳链球菌中的用途。
本发明还提供了一种无乳链球菌的检测方法，步骤如下：用本发明检测试纸的样品垫浸润待检样品，观察检测区和质控区是否显色，即可。
本发明还提供了一种无乳链球菌的检测方法，步骤如下：取待检样本，用前所述的检测试剂盒检测，即可。
优选地，所述待检样本为鱼体或者养殖水体。
本发明无乳链球菌单克隆抗体制备的胶体金快速检测试纸，其灵敏度高，特异性好，操作方便、快速，解决了现有无乳链球菌检测的问题，应用前景良好。
在本试纸成功研制之前，国际上尚无针对无乳链球菌的胶体金快速检测试纸的报道。从已有文献来看，研制该试纸所需要的生物材料非常难以制备和获取。因此，本试纸的成功研制在世界上尚属首次，处于世界领先水平。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1单抗的纯化结果
图2试纸的结构，1：PVC底板；2：硝酸纤维素膜；3：胶体金垫；4：吸水垫；5：检测区；6：质控区；7：样品垫；
图3 1：6×1010CFU/ml；2：6×109CFU/ml；3：6×108CFU/ml；4：6×107CFU/ml；5：6×106CFU/ml；C Line：质控线；T Line：检测线；
图4试纸条的特异性测试；
图5发病鱼组织及水体的检测结果。
具体实施方式
下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照试剂盒说明书选择。
实验材料：
细菌：无乳链球菌TW3用于克隆和制备重组抗原，无乳链球菌(ATCC51487)、无乳链球菌C918(牛源)、无乳链球菌TW7(鱼源)、无乳链球菌TW10(鱼源)、鮰爱德华氏菌、粪肠球菌、海豚链球菌、豚鼠气单胞菌、鼠伤寒沙门氏菌、金黄色葡萄球菌、霍乱弧菌、副溶血弧菌、嗜水气单胞菌、枯草芽孢杆菌、溶藻弧菌、阴沟肠杆菌、腐败希瓦氏菌、肺炎克雷伯氏菌、幽门螺杆菌、嗜麦芽寡养单胞菌、奇异变形杆菌、热带念珠菌、伤寒沙门氏菌、副伤寒沙门氏菌、粘质沙雷氏菌、大肠埃希氏菌、白色念珠菌、弗氏柠檬酸杆菌、绿脓杆菌、溶血葡萄球菌、鲍曼不动杆菌和淋病柰瑟氏菌由通威股份动物保健研究所保存。
实施例1 本发明检测试纸的制备
一、单克隆抗体的制备
1.1重组抗原的制备
1)基因克隆。用Promega公司的DNA提取试剂盒提取无乳链球菌TW3的基因组DNA，根据无乳链球菌sip基因(罗非鱼源，HQ878436.1，Genbank)，设计如表1所示的引物，上下游引物分别包含BamH I和Sal I限制性内切酶位点。采用设计的引物对扩增sip基因，扩增条件如下：DNA 94℃预变性5min后，设置程序94℃、30s，55℃、35s和72℃、78s，共30个循环；然后72℃延伸10min。
2)转化、表达与纯化。将扩增后的sip DNA连接到pET32a载体上，该载体含有编码六聚组氨酸的序列。用pET32a-sip转化大肠埃希氏菌BL21。转化子在含有100μg/mL卡那霉素的LB液体培养基中37℃培养。加入0.8mM异丙基-β-D-硫代半乳糖苷(IPTG)并于37℃再培养5h，以表达带六聚组氨酸标签的重组Sip(rSip)。超声震荡提取含有rSip的可溶部分，然后使用IMAC亲和层析柱进行纯化和洗脱，并使用二喹啉甲酸检测试剂盒(Sigma-Aldrich)测定蛋白浓度。
表1 用于表达sip基因的引物增子(～1302bp)
Figure PCTCN2016078451-appb-000001
3)电泳和免疫印迹。对纯化的rSip进行SDS-PAGE电泳(凝胶浓度12％)，考马斯亮蓝染色或免疫印迹来显示蛋白质条，同时使用针对无乳链球菌的多抗，以检测rSip的活性。
用蛋白rSip作为生产单抗的免疫原和ELISA检测中的抗原，rSip的氨基酸序列(SEQ ID NO.1)如下：
Figure PCTCN2016078451-appb-000002
1.2免疫程序
用经弗式完全佐剂(Sigma-Aldrich)乳化(乳化方法：将抗原和弗氏完全佐剂等体积混合，吸入一支注射器内，取下注射器针头，用软管连接另一支注射器，扎带固定好软管，轮流推动两支注射器，使混合液通过软管在注射器之间来回流动，从而达到乳化目的)的25μg,50μg，100μg和150μg rSip蛋白分别腹腔注射雌性BALB/c小鼠(8周龄)。4和8周后，用经弗式不完全佐剂(Sigma-Aldrich)乳化的相同抗原再次注射(乳化方法及注射剂量同前)小鼠。第10周，最后单独腹腔注射rSip蛋白(注射剂量同前)一次。
1.3杂交瘤产生和针对Sip蛋白的单抗生产
最后一次加强注射后三天，获取免疫小鼠脾细胞(分离方法：取高免BALB/c小鼠，拉颈处死后，于75％酒精中浸泡3～5min，无菌操作打开腹腔， 并小心取出脾脏至于平皿中，加入少量DMEM基础培养基漂洗2～3次，并仔细去除脾脏周围的结缔组织。然后在另一培养皿中倒入无血清将铜网盖于其上，取脾脏于铜网上，用研磨棒轻轻研磨，待细胞释放完全后，收集脾细胞，1000rpm离心10min)。根据Situ和Wu描述的方法：用50％(w/v)聚乙二醇4000(Sigma-Aldrich)，将脾细胞和骨髓瘤细胞S/P20以5:1的比例进行融合(细胞融合的步骤：S/P20细胞由成都中医药大学饶朝龙教授惠赠。融合步骤：取对数生长期的S/P20细胞与脾细胞按比例充分混匀，1000rpm离心10min，弃上清液，用手掌轻击离心瓶底，打散沉淀的细胞。将离心瓶置于40℃水浴中，在1min内边旋转边加入1ml PEG4000，再加入15ml无血清RPMI-1640培养基终止融合，在90s内加完，由慢到快，前5s加1ml。室温静置10min，1000rpm离心10min，弃上清，将含HAT和15％胎牛血清的RPMI-1640培养基加到细胞融合物中，悬浮细胞，分配到已有饲养细胞的96孔细胞培养板，置于37℃、5％CO2的温箱中培养)，得到杂交瘤细胞。
筛选步骤1：
将得到的杂交瘤细胞在次黄嘌呤-氨蝶呤-胸苷培养基中培养，以5μg/ml的rSip作为包被抗原，用ELISA检测培养上清中的抗体，以筛选杂交瘤细胞，得到ELISA鉴定为阳性的杂交瘤细胞。
筛选步骤2：
将ELISA鉴定为阳性的杂交瘤细胞于BALB/c小鼠腹水中增殖。收集腹水，使用Protein G Sepharose亲和层析柱(GE Healthcare Life Sciences)纯化(纯化方法：用注射器穿刺吸取小鼠腹水，在4℃，12000g条件下离心15min除去较大的凝块。将处理好的腹水用0.02M pH 7.0的PBS缓冲液稀释10倍，0.45μm滤膜过滤，然后上样流经0.02M pH 7.0的PBS缓冲液平衡好的蛋白G亲和层析柱。用0.1M pH 2.7的Gly-HCl为洗脱液进行洗脱，收集洗脱峰，并用1.0M pH 9.0的Tris-HCl调pH至约7.0，最后透析脱盐并冷冻干燥)腹水中的rSip单抗。
将纯化的单抗(浓度2mg/ml)与胶体金液体结合，室温(RT)放置5min。加入5％PEG后，立即8000rpm离心30min。于12ml离心管中收集红色沉淀，用胶体金保护剂重悬，并稀释至工作浓度(通常OD532＝30-50)。然后将溶液置于4℃。
用0.1M PBS将另一单抗稀释至2mg/ml。用0.1M PBS将羊抗鼠IgG稀释至1.0mg/ml。前者用于包被NC膜上的检测区，后者用于质控区。然后将 膜在37℃下保持24h。用BB缓冲液(Artron BioResearch Inc)封闭后，用WB缓冲液洗膜，然后室温干燥。
试纸条组装有样品垫、结合垫、反应膜和吸附垫。用自动切条机将试纸切成3mm小条。
将所有结合的-单抗与包被的-单抗配对，用于试纸条制备。将试纸条的样品垫插入无乳链球菌样品中，直至NC膜的一半浸入到液体中。室温下，3-10min后观察到质控区和检测区的形成。筛选对无乳链球菌显示出最强的阳性信号，对其它受试细菌显示出阴性结果的配对抗体。
细胞融合后共获得84个阳性细胞系。纯化所有84个单抗并用作试纸条组装的包被抗体/结合抗体。经过多次配对测试之后，找到NC7b-CE12配对显示出对无乳链球菌有最强信号，对其它细菌无信号。
将分别表达单抗NC7b和单抗CE12的杂交瘤细胞株，分别命名为杂交瘤细胞株TWDB-WR-1和杂交瘤细胞株TWDB-WR-2，并于2015年10月21日保藏在中国典型培养物中心(CCTCC)，保藏号分别为CCTCC NO：C2015178和CCTCC NO：C2015177。
2、单抗制备
取杂交瘤细胞株TWDB-WR-1和杂交瘤细胞株TWDB-WR-2分别按照如下方法制备单抗NC7b和单抗CE12：
取前述杂交瘤细胞株于BALB/c小鼠腹水中增殖，具体是将灭菌液体石蜡腹腔注射8周龄的BALB/c小鼠，0.5ml/只。7天后，将前述两株杂交瘤细胞株分别用无血清RPMI-1640培养基悬起，1000rpm离心5min，弃上清，洗去培养基中的血清，再用适量无血清RPMI-1640培养基重悬细胞沉淀，分别注射入小鼠腹腔，每只0.5ml(含约106个杂交瘤细胞。收集腹水(7天后采取腹部明显鼓起小鼠的腹水)，使用Protein G Sepharose亲和层析柱(GE Healthcare Life Sciences)纯化(纯化的具体步骤：用注射器穿刺吸取小鼠腹水，在4℃，12000g条件下离心15min除去较大的凝块。将处理好的腹水用0.02M pH 7.0的PBS缓冲液稀释10倍，0.45μm滤膜过滤，然后上样流经0.02M pH 7.0的PBS缓冲液平衡好的蛋白G亲和层析柱。用0.1M pH2.7的Gly-HCl为洗脱液进行洗脱，收集洗脱峰，并用1.0M pH 9.0的Tris-HCl调pH至约7.0，最后透析脱盐并冷冻干燥)腹水中的rSip单抗。
纯化结果见图1所示，可以看出，本发明获得了纯品的单抗NC7b和单抗CE12。
二、检测试纸的制备
本发明试纸条为双抗体夹心法试纸条，试纸结构图图2所示：
胶体金制备：主要采用柠檬酸三钠还原法制备胶体金。在10mL体系氯金酸溶液中加入150μl柠檬酸钠，肉眼观察胶体金颗粒颜色均一，呈酒红色，有明显光晕，经电镜观察胶体金颗粒大小一致，颗粒直径在20nm左右。经紫外扫描金颗粒，其峰宽较窄，表明胶体金溶液分散性良好。
金标抗体的制备方法：取单抗CE12(步骤一制备)，与胶体金液体结合，室温(RT)放置5min。加入5％PEG后，立即8000rpm离心30min。于12ml离心管中收集红色沉淀，用胶体金保护剂重悬，并稀释至工作浓度(OD532＝30-50)，4℃保存备用)。
将标记好的金标抗体包被于玻璃纤维膜上，制成胶体金垫：取金标抗体溶液加入到喷涂机的喷头储存瓶中，喷涂玻璃纤维膜，烘干，即为胶体金垫。
试纸组装(结构如图2所示)：PVC底板(1)上粘贴的硝酸纤维素膜(2)，样品垫(7)通过胶体金垫(3)与硝酸纤维素膜(2)一端连接，硝酸纤维素膜(2)另一端搭接有吸水垫(4)，所述硝酸纤维素膜(2)上靠近胶体金垫(3)的一侧包被有本发明单抗NC7b(步骤一制备)作为检测区(5)，靠近吸水垫(4)的一侧包被有羊抗鼠IgG作为质控区(6)。
在胶体金垫上包被抗体的方法：取抗体，制成抗体溶液，加入到喷涂机的喷头储存瓶中，喷涂试纸，烘干，即可。
实施例2 本发明检测试纸的性质检测
1性质检测
1.1试纸条灵敏度测试
将无乳链球菌倍比稀释为6×1010CFU/ml至6×106CFU/ml，然后用试纸条(即本发明实施例1建立的检测试纸)测试以评估其最小检测数量。
1.2试纸条特异性测试
用试纸条测试无乳链球菌(ATCC 51487)、无乳链球菌C918(牛源)、鮰爱德华氏菌、粪肠球菌、海豚链球菌、豚鼠气单胞菌、鼠伤寒沙门氏菌、金黄色葡萄球菌、霍乱弧菌、副溶血弧菌、嗜水气单胞菌、枯草芽孢杆菌、溶藻弧菌、阴沟肠杆菌、腐败希瓦氏菌、肺炎克雷伯氏菌、幽门螺杆菌、嗜麦芽寡养单胞菌、奇异变形杆菌、热带念珠菌、伤寒沙门氏菌、副伤寒沙门 氏菌、粘质沙雷氏菌、大肠埃希氏菌、白色念珠菌、弗氏柠檬酸杆菌、绿脓杆菌、溶血葡萄球菌、鲍曼不动杆菌和淋病柰瑟氏菌。
1.3试纸条重复性测试
分别用试纸条测试三个无乳链球菌样品和三个阴性样品，每个样品重复测试十次。
1.4实际样品测试
采集发病罗非鱼，解剖并取豌豆大小的待检组织(肝或脑)于1.5ml Ep管中，捣碎后，加入1ml生理盐水，混匀后进行测试，同时无菌操作分离肝脏内的细菌，纯化后使用PCR体系进行检测，比较二者的检测结果。
采用本发明试纸的测试方法：用本发明检测试纸的样品垫浸润待检样品，观察检测区和质控区是否显色，即可。其中，若试纸的检测区5和质控区6位置上均出现红色条带，则待检样品为检测阳性；若仅在质控区6位置上出现红色条带，而检测区5位置上无红色条带，则待检样品为检测阴性；若试纸条的质控区6位置上未出现红色条带，无论检测区位置上是否出现红色条带，检测结果都无效。
2检测结果
2.1灵敏度测试结果
将无乳链球菌培养液10倍稀释至6×1010CFU/ml～6×106CFU/ml，以测试试纸条的灵敏度。当浓度为6×107CFU/ml时，可见微弱的T线，但当浓度继续降至6×106CFU/ml时，T线不会出现(图3)。图3显示该配对抗体的检测限为6×107CFU/ml。
表2 无乳链球菌试纸条的检测限
无乳链球菌 血清型 分离源 检测限
TW3 Ia 鱼 6×107CFU/ml
TW6 Ia 鱼 1.1×107CFU/ml
TW7 Ia 鱼 1.3×107CFU/ml
TW9 Ia 鱼 1.1×106CFU/ml
TW10 Ia 鱼 1×107CFU/ml
TW31 II 牛源 阴性
2.2特异性测试结果
两个配对的快速检测试纸条对无乳链球菌具有高度特异性，与其它测试细菌无交叉反应(图4和表3)。
表3 无乳链球菌试纸条的特异性测试
Figure PCTCN2016078451-appb-000003
2.3重复性测试结果
阴阳性符合率均为100％(表4)。
表4 重复性测试结果
Figure PCTCN2016078451-appb-000004
2.4实际样品的检测
使用无乳链球菌检测试制对发病罗非鱼及发病池塘的水体进行了检测，结果表明，发病鱼体内含有大量的无乳链球菌，而水体中没有检测到无乳链球菌(图5)，与PCR的检测结果一致(结果未提供)，提示无乳链球菌在鱼体内得到了增殖，从而引发疾病。
试验结果说明，本发明无乳链球菌检测试纸的灵敏度高、特异性强、重复性好，可以准确检测实际样品。

Claims (16)

  1. 一种检测无乳链球菌的胶体金免疫层析试纸，其特征在于：采用的两种无乳链球菌抗体为中国典型培养物中心保藏的保藏号：CCTCC NO：C2015177的细胞株分泌的抗体以及中国典型培养物中心保藏的保藏号：CCTCC NO：C2015178的细胞株分泌的抗体。
  2. 根据权利要求1所述的检测试纸，其特征在于：所述中国典型培养物中心保藏的保藏号：CCTCC NO：C2015177的细胞株分泌的抗体用于制备金标抗体；中国典型培养物中心保藏的保藏号：CCTCC NO：C2015178的细胞株分泌的抗体用于包被硝酸纤维素膜，形成检测区。
  3. 根据权利要求1所述的检测试纸，其特征在于：所述抗体的制备方法如下：
    (1)分别取保藏号：CCTCC NO：C2015177的细胞株和保藏号：CCTCC NO：C2015178的杂交瘤细胞株，注射入BALB/c小鼠腹水中增殖；
    (2)收集腹水，纯化，即可。
  4. 根据权利要求3所述的检测试纸，其特征在于：步骤(2)中，所述纯化是采用Protein G Sepharose亲和层析柱纯化。
  5. 根据权利要求1～4任意一项所述的检测试纸，其特征在于：所述试纸的结构如下：包括PVC底板(1)和设于其上的硝酸纤维素膜(2)，样品垫(7)通过胶体金垫(3)与硝酸纤维素膜(2)一端连接，硝酸纤维素膜(2)另一端搭接有吸水垫(4)，所述硝酸纤维素膜(2)上靠近胶体金垫(3)的一侧设有检测区(5)，靠近吸水垫(4)的一侧设有质控区(6)。
  6. 根据权利要求5所述的检测试纸，其特征在于：所述样品垫(7)搭接在胶体金垫(3)一端上表面，胶体金垫(3)另一端搭接在硝酸纤维素膜(2)上表面。
  7. 根据权利要求5所述的检测试纸，其特征在于：所述质控区(6)上包被有羊抗鼠的多克隆抗体(10)。
  8. 一种制备权利要求1～7任意一项所述的检测试纸的方法，其特征在于：步骤如下：
    a、取中国典型培养物中心保藏的保藏号：CCTCC NO：C2015177的细胞株分泌的抗体，与胶体金混合，制备金标抗体，喷涂于玻璃纤维膜上，制得胶体金垫；
    b、取硝酸纤维素膜，粘贴于PVC底板(1)上，将样品垫通过胶体金垫 与硝酸纤维素膜的一端连接，再在硝酸纤维素膜的另一端搭接吸水垫；
    c、再在硝酸纤维素膜靠近胶体金垫的一侧喷涂取中国典型培养物中心保藏的保藏号：CCTCC NO：C2015178的细胞株包被的抗体作为检测区，在靠近吸水垫的一侧包被羊抗鼠IgG作为质控区。
  9. 一种无乳链球菌的免疫学检测试剂盒，其特征在于：它包括如下两种无乳链球菌单克隆抗体：采用的两种无乳链球菌抗体为中国典型培养物中心保藏的保藏号：CCTCC NO：C2015177的细胞株分泌的抗体以及中国典型培养物中心保藏的保藏号：CCTCC NO：C2015178的细胞株分泌的抗体。
  10. 根据权利要求9所述的检测试剂盒，其特征在于：所述抗体的制备方法如下：
    (1)分别取保藏号：CCTCC NO：C2015177的细胞株和保藏号：CCTCC NO：C2015178的杂交瘤细胞株，注射入BALB/c小鼠腹水中增殖；
    (2)收集腹水，纯化，即可。
  11. 根据权利要求10所述的检测试剂盒，其特征在于：步骤(2)中，所述纯化是采用Protein G Sepharose亲和层析柱纯化。
  12. 根据权利要求9～11任意一项所述的检测试剂盒，其特征在于：所述试剂盒还包括免疫荧光检测用试剂、放射免疫检测用试剂、酶联免疫吸附检测用试剂或免疫金胶体检测用试剂。
  13. 权利要求1～7任意一项所述的检测试纸以及权利要求9～12任意一项所述的检测试剂盒在制备检测无乳链球菌的试剂中的用途。
  14. 一种无乳链球菌的检测方法，其特征在于：步骤如下：用权利要求1～7任意一项所述的检测试纸的样品垫浸润待检样品，观察检测区和质控区是否显色，即可。
  15. 一种无乳链球菌的检测方法，其特征在于：步骤如下：取待检样本，用权利要求9～12任意一项所述的检测试剂盒检测，即可。
  16. 根据权利要求14或者15所述的检测方法，其特征在于：所述待检样本为鱼体或者养殖水体。
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107290527A (zh) * 2017-07-31 2017-10-24 南京医科大学第附属医院 用于检测乙酰胆碱酯酶的胶体金层析快速诊断试纸条及其制备方法
CN110806477A (zh) * 2018-08-06 2020-02-18 国家纳米科学中心 一种病原菌检测试纸条、传感器及其应用
CN111398587A (zh) * 2020-04-02 2020-07-10 安徽科技学院 一种检测宫颈癌的胶体金侧向层析试纸条及其制备方法

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105372422B (zh) * 2015-11-06 2017-05-31 通威股份有限公司 无乳链球菌的胶体金快速检测试纸
CN107656063A (zh) * 2017-03-29 2018-02-02 广西大学 罗非鱼无乳链球菌免疫层析快速检测试纸条及其制备方法
CN107942060A (zh) * 2017-11-16 2018-04-20 广西大学 一种利用免疫胶体金渗滤法检测罗非鱼无乳链球菌抗体的方法
CN111929436A (zh) * 2020-10-09 2020-11-13 中南大学湘雅医院 一种用于冷冻电镜的免疫金标记蛋白质方法
CN112305223B (zh) * 2020-11-13 2022-08-05 福建农林大学 一种检测细交链孢菌酮酸的核壳型金铂合金纳米免疫层析试纸

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2533503A1 (en) * 2003-07-23 2005-02-03 Universita' Degli Studi Di Pavia Antibodies to the fbsa protein of streptococcus agalactiae and their use in treating or preventing infections
CN203224497U (zh) * 2013-04-27 2013-10-02 通威股份有限公司 一种细菌的胶体金检测试纸
CN104198741A (zh) * 2014-07-23 2014-12-10 福建省淡水水产研究所 罗非鱼无乳链球菌IgM抗体捕获ELISA检测试剂盒
WO2015093546A1 (ja) * 2013-12-18 2015-06-25 旭化成株式会社 乳汁中のストレプトコッカス属の菌を検出する方法
CN105274062A (zh) * 2015-11-06 2016-01-27 通威股份有限公司 无乳链球菌单克隆抗体及其制备方法和用途
CN105372422A (zh) * 2015-11-06 2016-03-02 通威股份有限公司 无乳链球菌的胶体金快速检测试纸

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104142396A (zh) * 2013-05-10 2014-11-12 北京东方冷翠国际技术有限公司 一种肠出血性大肠杆菌快速检测方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2533503A1 (en) * 2003-07-23 2005-02-03 Universita' Degli Studi Di Pavia Antibodies to the fbsa protein of streptococcus agalactiae and their use in treating or preventing infections
CN203224497U (zh) * 2013-04-27 2013-10-02 通威股份有限公司 一种细菌的胶体金检测试纸
WO2015093546A1 (ja) * 2013-12-18 2015-06-25 旭化成株式会社 乳汁中のストレプトコッカス属の菌を検出する方法
CN104198741A (zh) * 2014-07-23 2014-12-10 福建省淡水水产研究所 罗非鱼无乳链球菌IgM抗体捕获ELISA检测试剂盒
CN105274062A (zh) * 2015-11-06 2016-01-27 通威股份有限公司 无乳链球菌单克隆抗体及其制备方法和用途
CN105372422A (zh) * 2015-11-06 2016-03-02 通威股份有限公司 无乳链球菌的胶体金快速检测试纸

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BERNARD, R.B. ET AL.: "Identification of Group B Streptococcal Sip Protein, Which Elicits Cross-Protective Immunity", INFECTION AND IMMUNITY, vol. 68, no. 10, 30 October 2000 (2000-10-30), pages 5610 - 5618, XP002666702 *
TATSUYA, N. ET AL.: "Colloidal gold-based immunochromatographic strip test compromising optimised combinations of anti-S. suis capsular polysaccharide polyclonal antibodies for detection of Streptococcus suis", BIOSENSORS AND BIOELECTRONICS, vol. 60, 15 October 2014 (2014-10-15), pages 175 - 179, XP055378743 *
ZHANG, XINYAN: "Establishment of DAS-ELISA Detection for Streptococcus Agalaciate in Tilapia", FUJIAN JOURNAL OF AGRICULTURAL SCIENCES, vol. 29, no. 6, 15 June 2014 (2014-06-15), pages 570 - 574, XP055378742 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107290527A (zh) * 2017-07-31 2017-10-24 南京医科大学第附属医院 用于检测乙酰胆碱酯酶的胶体金层析快速诊断试纸条及其制备方法
CN110806477A (zh) * 2018-08-06 2020-02-18 国家纳米科学中心 一种病原菌检测试纸条、传感器及其应用
CN111398587A (zh) * 2020-04-02 2020-07-10 安徽科技学院 一种检测宫颈癌的胶体金侧向层析试纸条及其制备方法
CN111398587B (zh) * 2020-04-02 2023-02-28 安徽科技学院 一种检测宫颈癌的胶体金侧向层析试纸条及其制备方法

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