WO2015054940A1 - 一种crp/pct联合诊断试纸及其制备方法 - Google Patents

一种crp/pct联合诊断试纸及其制备方法 Download PDF

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WO2015054940A1
WO2015054940A1 PCT/CN2013/086885 CN2013086885W WO2015054940A1 WO 2015054940 A1 WO2015054940 A1 WO 2015054940A1 CN 2013086885 W CN2013086885 W CN 2013086885W WO 2015054940 A1 WO2015054940 A1 WO 2015054940A1
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antibody
pct
crp
labeled
solution
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PCT/CN2013/086885
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French (fr)
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张二盈
章国建
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深圳市大爱医疗科技有限公司
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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
    • G01N33/531—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
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4737—C-reactive protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • the invention relates to the technical field of biomedical detection, in particular to a CRP/PCT combined diagnostic test paper and a preparation method thereof.
  • C-reactive protein C-reactive
  • PCT procalcitonin
  • Fluorescence immunochromatography is an immunoassay based on fluorescence chromatography technology and antigen-antibody-specific immune response.
  • Fluorescent immunochromatographic test strips generally include sample mats that are sequentially placed on the bottom plate and closely overlapped. The bonding pad, the reaction film and the water absorption pad are arranged in the plastic card, and the plastic card is provided with a sample hole and a display window respectively matched with the sample pad and the reaction film.
  • CRP is an acute phase reaction protein synthesized by the liver to form a complex with the S. pneumoniae C polysaccharide. It is joined by five identical subunits in a non-covalent manner. CRP not only has important clinical application prospects for infectious diseases, disease course detection and prognosis evaluation of gestational diabetes, antibiotic efficacy, but also one of the most important predictors of cardiovascular disease. Normal human serum has very little CRP content. In general, serum CRP levels in newborns are less than 2 mg/L. Greater than this value is related to the severity of bacterial infection; serum CRP levels in children and adults are less than 10 mg/L; 10-99 mg/L suggest focal or superficial infections, greater than Or equal to 100mg / L to indicate serious infections such as sepsis or invasive infection.
  • CRP began to rise 6-8h after infection, peaked at 24-48h, several hundred times or even thousands of times higher than normal, and the increase was positively correlated with the degree of infection. After the disease is cured, its content drops rapidly and returns to normal within a week.
  • CRP is generally used as a preferred indicator for identifying bacterial or viral infections, for the diagnosis and monitoring of autoimmune and infectious diseases, and for the observation of antibiotic efficacy.
  • Low levels of CRP also known as hypersensitive C-reactive protein (hs-CRP).
  • hs-CRP hypersensitive C-reactive protein
  • hs-CRP ⁇ 1mg / L is a low risk; 1-3mg / L is a moderate risk; greater than 3mg / L is a high risk.
  • PCT is a precursor to calcitonin. Under normal circumstances, calcitonin is secreted only by C cells of the thyroid gland after being stimulated by hormones, and its concentration is usually less than 0.1 ng/mL. In the case of pro-inflammatory stimuli, especially when infected by bacteria, PCT is produced by a large variety of cells. When the PCT concentration is 0.1-0.25 ng/mL, it is unlikely to infect bacteria; 0.25-0.5 ng/mL may infect bacteria; more than 0.5 ng/mL is very likely to infect bacteria, requiring antibiotic treatment.
  • PCT can observe an increase in PCT within 3-6 hours of infection stimulation, and the PCT is increasing with the increase of sensibility.
  • CRP can occur in both viral and bacterial diseases, and PCT can only occur in bacterial infections. Therefore, PCT as a diagnostic indicator has the characteristics of rapidity and specificity.
  • Combined application of CRP and PCT will be able to more accurately determine the degree of inflammation infection.
  • the detection of hs-CRP should generally exclude inflammatory infections. Therefore, the combination of CRP and PCT will be very important. Therefore, the combined application of PCT and CRP will be more widely used, more convenient, and more accurate in diagnosis than when they are used alone.
  • a quantum dot is an aggregate composed of a certain number of actual atoms, and a semiconductor compound having a three-dimensional size of less than 100 nm. It has many advantages such as high luminous intensity, wide excitation spectrum, narrow emission spectrum, long fluorescence lifetime, multi-functional surface modification and good stability. It has the potential to replace traditional organic fluorescent dyes in the field of fluorescence detection and has become a new generation of bio-fluorescence. Mark.
  • the technical problem solved by the invention is based on the advantages of the combined diagnosis of CRP and PCT, and the fluorescence characteristics of the quantum dots, and provides a PCT/CRP combined diagnostic test paper with high sensitivity, fastness and accuracy.
  • the present invention adopts the following technical solutions:
  • a CRP/PCT joint diagnostic test paper comprises a sample pad, a bonding pad, a reaction film and an absorbent pad which are arranged on the bottom plate and are closely connected in sequence.
  • the bottom plate is arranged in a plastic card, and an anti-seepage is arranged longitudinally in the middle of the bottom plate.
  • a water-repellent strip separating the sample pad, the bonding pad, the reaction film and the absorbent pad into a first sample pad, a second sample pad, a first bonding pad, a second bonding pad, and a first reaction film a second reaction membrane, a first absorbent pad and a second absorbent pad;
  • the first binding pad is immobilized with a labeled CRP antibody I
  • the second binding pad is immobilized with a labeled PCT antibody I
  • the first reaction membrane and the first The two reaction membranes are provided with a quantitative belt and a quality control belt.
  • the labeled CRP antibody I is a quantum dot-labeled CRP antibody I
  • the labeled PCT antibody I is a quantum dot-labeled PCT antibody I.
  • the quantum dot-labeled CRP antibody I has a different emission wavelength than the quantum dot-labeled PCT antibody I.
  • a first quantitative band and a first control band are disposed on the first reaction membrane, and the first quantitative band is immobilized with a CRP antibody II having a different antigen binding site from the labeled CRP antibody I, the first The secondary control of the labeled CRP antibody I was immobilized on the control strip.
  • the second reaction membrane is provided with a second quantitative band and a second control band, and the second quantitative band is immobilized with PCT antibody II having a different antigen binding site from the labeled PCT antibody I, the first quality control The secondary antibody with the labeled PCT antibody I immobilized.
  • the distance between the quantitative tape and the quality control tape is 3-5 mm.
  • the bottom plate is a black bottom plate.
  • the sample pad is a glass chromatography membrane.
  • the test paper has a two-slot plastic card that is disposed in a two-slot plastic card.
  • the plastic card of the double-insertion slot is provided with two parallel sample loading holes and two parallel display windows.
  • the preparation method of the above CRP/PCT joint diagnostic test paper comprises the following steps:
  • a quantum dot-labeled CRP antibody I solution having different emission wavelengths and a quantum dot-labeled PCT antibody I solution were sprayed onto the first binding pad and the second bonding pad, respectively, and air-dried at room temperature, and stored at 4 ° C for use.
  • the quantum dot-labeled CRP antibody I/PCT antibody I solution is added to each of 100 mL, 1.5-2.5 mg/mL of quantum dot-labeled CRP antibody I/PCT antibody I, and 2-5 g of sucrose, 0.5-1 g, respectively.
  • BSA a solution of 0.05-0.1 g Tween.
  • the CRP/PCT test strip obtained in the step c is inserted into the double-slot plastic card.
  • the concentration of the phosphate buffer having a pH of 7.2 to 7.5 was 10 mmol/L.
  • the ultrafiltration was concentrated to centrifugation for 6-8 min using a 100 K ultrafiltration tube at 20-25 ° C and 5000-7000 r/min.
  • the quantum dots are carboxyl water-soluble quantum dots; the carboxyl water-soluble quantum dots are quantum dots formed by core-shell quantum dots or a single compound.
  • the core-shell type quantum dots are quantum dots formed by ZnS/CdSe or ZnS/CdTe.
  • the single compound is any one of the following compounds: a compound formed of a Group IIIA element and a Group VA element, a compound formed of a Group IIA element and a Group VIA element, a Group IIB element, and a Group VIA element. a compound consisting of a compound, a Group IVA element, and a Group IVA element compound, a Group IVA element, and a Group VIA element.
  • the single compound is GaSb, InAs, InP, InGaAs, InAlAs, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, ZnS, ZnSe, ZnTe, CdS, CdSe, Any of CdTe, SiC, SiGe, SiSe, SiTe, and SiS.
  • EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide), respectively, and EDC And the NHS is directly added to the quantum dot solution obtained in the step (1), mixed with a shaker for 5-8 times, and then reacted at 23-25 ° C for 50-70 min to obtain an activated quantum dot; the EDC, NHS and the quantum dot molar The ratio is (20000-25000): 5000:1.
  • the molar ratio of EDC, NHS and quantum dots is 25000:5000:1.
  • EDC and NHS are weighed at 0-4 ° C and air humidity is less than or equal to 30%, and are quickly added to the reaction liquid. More preferably, EDC and NHS are weighed in an ice box at 0-4 °C.
  • the activated quantum dots are stored in a buffer and concentrated by ultrafiltration for use.
  • the activated quantum dot solution obtained in the step (3) is uniformly mixed with the CRP antibody I or PCT antibody I solution obtained in the step (4), and reacted at 23-25 ° C for 5-7 hours under shaking to obtain a CRP antibody.
  • I or PCT antibody I-quantum dot conjugate reaction solution the molar ratio of activated quantum dots to CRP antibody I or PCT antibody I is 1:8-10.
  • the molar ratio of activated quantum dots to CRP antibody I or PCT antibody I is 1:10, and the concentration of activated quantum dots is 0.8 ⁇ mol/L.
  • CRP antibody I or PCT antibody I-quantum dot conjugate solution and CRP antibody I or PCT antibody I recovery solution are obtained by the exclusion reaction step (5); CRP antibody I or PCT antibody
  • the I-quantum dot conjugate solution was concentrated by ultrafiltration and added to the blocking solution and stored at 4 ° C; the CRP antibody I or PCT antibody I recovery solution was concentrated by ultrafiltration and recovered for reuse.
  • the exclusion chromatography column used in the exclusion chromatography was superdex 200 or Sephacryl 300, the flow rate was 1.5 ml/min, and the column length was 40-60 cm.
  • the blocking solution was a Gly blocking solution, a Gly concentration of 1 mg/mL, a solvent of 50 mmol/L, and a pH of 8.3-8.5 borate buffer.
  • the invention has the beneficial effects that the invention integrates CRP detection and PCT detection into the same test strip, and can simultaneously detect the concentration of CRP and PCT, which not only improves the detection efficiency, but also has the CRP detection and Advantages of PCT detection.
  • the quantum dot-labeled antibody is immobilized on the binding pad, and the sensitivity and accuracy of the test paper are improved by utilizing the superior fluorescence characteristics of the quantum dot.
  • the method for preparing the quantum dot-labeled CRP antibody I or the PCT antibody I of the invention has high labeling efficiency of the quantum dots, and the prepared labeled antibody has high stability and high activity, thereby further improving the sensitivity and accuracy of the test paper.
  • Figure 1 is a schematic view showing the structure of a PCT/CRP joint diagnostic test paper without a double-slot plastic card
  • FIG. 2 is a schematic structural view of a PCT/CRP joint diagnostic test paper provided with a two-slot plastic card;
  • Example 3 is a capillary electrophoresis identification of quantum dot labeling efficiency of Example 1 and Example 2;
  • Example 4 is a fluorescence spectrum diagram of the PCT-QDs prepared in Example 2 after being placed for different periods of time.
  • the carboxyl group water-soluble quantum dot CdTe/Zn with emission wavelength of 525 nm was selected for CRP antibody I labeling, which was recorded as CdTe/Zn-525.
  • the quantum dot CdTe/Zn with emission wavelength of 650 nm was selected for labeling of PCT antibody I, which was recorded as CdTe/. Zn-650.
  • the pH of the quantum dot solution was checked with a micro pH meter and the pH was adjusted to 7.3. Weigh 2mmol in ice box with humidity not exceeding 30% and 0-4°C EDC and 0.5mmol Two parts of NHS were quickly added to the two quantum dot solutions. The vortex oscillator was shaken uniformly and placed on a shaker for 60 min. The reaction temperature was 24 ° C to activate CdTe/Zn-525 and activated CdTe/Zn-650, respectively.
  • the CdTe/Zn-525 solution was mixed with the CRP antibody I solution, and the CdTe/Zn-650 solution was mixed with the PCT antibody I solution, mixed and reacted on a shaker for 6 h, respectively, and the reaction temperature was 23-25 ° C to obtain a CRP antibody I-CdTe. /Zn-525 complex reaction solution and PCT antibody I-CdTe/Zn-650 complex reaction solution.
  • the coupling of CdTe/Zn-650 with PCT antibody I was detected by capillary electrophoresis. The results are shown in Figure 3.
  • the CRP antibody I-CdTe/Zn-525 complex and the PCT antibody I-CdTe/Zn-650 complex were separated by exclusion chromatography, and the complex was concentrated to 200 ⁇ L with a 100K ultrafiltration tube. After the concentration of the complex, a Gly blocking solution having a Gly (glycine) concentration of 1 mg/mL was added and stored at 4 ° C until use.
  • the exclusion chromatography column was superdex200 with a flow rate of 1.5 mL/min and a gel length of 40 cm.
  • the first binding pad spray solution is added to 2-5 g of sucrose and 0.5-1 g of the quantum dot-labeled CRP antibody I per 100 mL, 1.5-2.5 mg/mL, respectively.
  • BSA 0.05-0.1g Tween solution
  • the second binding pad spray solution is added to each of the 100 mL, 1.5-2.5 mg/mL quantum dot-labeled PCT antibody I, respectively, 2-5 g of sucrose, 0.5-1 g BSA, a solution of 0.05-0.1 g Tween.
  • the first bonding pad spraying liquid is evenly sprayed on the first bonding pad 21, and the second bonding pad spraying liquid is evenly sprayed on the second bonding pad 22, air-dried at room temperature, and kept at 4 ° C for use.
  • a second antibody solution labeled with CRP antibody I is drawn on the first reaction membrane and blown dry to form a first control line, and a CRP antibody II solution having a different antigen binding site from the labeled CRP antibody I is used on the first reaction membrane.
  • the point PCT antibody II solution was drawn on the second reaction membrane and blown dry to form a second quantitative line;
  • the concentration of the secondary antibody solution labeled with CRP antibody I was 1.2 mg/ml; the concentration of the CRP antibody II solution was 0.8 mg/ml; the concentration of the secondary antibody solution labeled with PCT antibody I was 1.2 mg/ml; the concentration of the PCT antibody II solution It is 1.5 mg/ml.
  • the bottom plate of the test paper is a black bottom plate, and a water-proof strip 5 is longitudinally disposed in the middle of the bottom plate, and the water-proof strip 5 divides the bottom plate into two halves.
  • the first sample pad 11 and the second sample pad 12 are glass chromatography films.
  • the first sample pad 11, the first bonding pad 21, the first reaction film 31, and the first water absorbing pad 41 are sequentially disposed on one half of the bottom plate and closely overlapped, and the second sample pad 12, the second bonding pad 22, and the second
  • the second reaction film 32 and the second water absorption pad 42 are sequentially disposed on the other half of the bottom plate and are closely overlapped.
  • the bottom plate to which the sample pad, the bonding pad, the reaction film, and the absorbent pad are fixed is inserted into the double-slot plastic card.
  • the two-slot plastic card 6 is provided with two juxtaposed sample holes 62 and two juxtaposed display windows 61.
  • the two sample holes 62 are located above the first sample pad 11 and the second sample pad 12, respectively, and the sample is dropped onto the sample pad through the sample hole 62.
  • the two display windows 61 are respectively located above the first reaction film 31 and the second reaction film 32, and respectively observe and measure the first quality control tape 312, the second quality control tape 322, the first quantitative tape 311 and the first through the display window 61. The detection of the second quantitative band 321 .
  • Example 2 differs from Example 1 in that the process parameters for preparing the quantum dot-labeled PCT antibody I are different.
  • the above procedure was repeated three times so that the volume of the last activated CdTe/ZnS-650 solution did not exceed 200 ⁇ L.
  • the pH of the activated CdTe/ZnS-650 solution was measured with a micro pH meter and the pH was adjusted to 8.3.
  • the pH of the PCT antibody solution was measured with a micro pH meter and the pH was adjusted to 8.3.
  • the activated CdTe/ZnS-650 solution was mixed with the PCT antibody I solution, mixed and placed on a shaker for 5 h, and the reaction temperature was 23 ° C to obtain a PCT antibody I-CdTe/ZnS-650 reaction solution.
  • the coupling of quantum dots and PCT antibody I was detected by capillary electrophoresis, as shown in FIG.
  • the PCT antibody I-CdTe/Zn-650 complex was obtained by separating the reaction solution by exclusion chromatography, and the PCT antibody I-CdTe/Zn-650 complex was concentrated to 200 ⁇ L with a 100K ultrafiltration tube, and the concentration of the complex was measured and then added to Gly.
  • Glycine Gly blocking solution at a concentration of 1 mg/mL, and stored at 4 ° C, and set aside.
  • the fluorescence spectra of the purified PCT antibody I-CdTe/Zn-650 complex prepared in this example were measured by fluorescence spectrophotometer for 5 min, 1 h, 24 h, 3 d, 10 d and 30 d, respectively.
  • the detection results are shown in Fig. 4.
  • 1 is the curve of QDs; 2 is QDs-PCT, 5min curve; 3 is QDs-PCT, 1h curve; 4 is QDs-PCT, 3h curve; 5 is QDs-PCT, 10d curve; 6 is QDs-PCT, 30d curve.
  • the slight peaks of fluorescence of PCT-QDs placed at different time periods have a tendency to decrease, but the change is very slow, indicating that the resulting PCT-QDs are relatively stable.

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Abstract

一种CRP/PCT联合诊断试纸及其制备方法，其中试纸的底板中部纵向设有一防渗水胶条（5），并在防渗水胶条（5）两侧的底板上分别设样品垫（11，12）、结合垫（21，22）、反应膜（31，32）和吸水垫（41，42）；一结合垫（21）上固定有标记CRP抗体I，另一结合垫（22）上固定有PCT抗体I，两反应膜（31，32）上均设有定量带（311，321）和质控带（312，322）。将CRP检测和PCT检测整合于同一检测试纸中，可同时检测CRP和PCT的浓度，不仅提高了检测效率，还同时具备CRP检测和PCT检测的优点。其中量子点标记的CPR和PCT抗体的制备方法、量子点标记的效率高，制备的标记抗体的稳定性高、活性高，将其分别固定于结合垫上可提高试纸的灵敏度和准确性。

Description

一种CRP/PCT联合诊断试纸及其制备方法
技术领域
　　　本发明涉及生物医学检测技术领域，尤其涉及一种CRP/PCT联合诊断试纸及其制备方法。
背景技术
　　　在急诊治疗和重症监护过程中，快速而准确的评估细菌脓毒症严重程度是医师必须面临的挑战。C-反应蛋白（C-reactive protein，CRP）和降钙素原（Procalcitonin，PCT）的引入，为早期感染诊断和治疗反应评估提供了有效的方法。荧光免疫层析是一种建立在荧光层析技术和抗原-抗体特异性免疫反应基础上的一种免疫检测技术，荧光免疫层析试纸一般包括依次设于底板上且紧密搭接的样品垫、结合垫、反应膜和吸水垫，底板设于塑料卡内，塑料卡上设有分别与样品垫和反应膜配合的加样孔和显示窗口。
　　　CRP是由肝脏合成的一种能与肺炎链球菌C多糖体反应形成复合物的一种急性时相反应蛋白。它由5个相同的亚基单位以非共价方式联结。CRP不仅对感染性疾病、妊娠期糖尿病的病程检测及预后判断评估、抗生素疗效等具有重要的临床应用前景，同时也是心血管疾病最重要的预测因子之一。正常人血清中CRP含量极微。一般新生儿血清CRP水平小于2mg/L，大于此值即与细菌感染的严重程度有关；儿童和成人血清CRP水平小于10mg/L；10-99mg/L提示局灶性或浅表性感染，大于或等于100mg/L提示败血症或侵袭性感染等严重感染。CRP在感染发生后6-8h开始升高，24-48h达到高峰，比正常值高几百倍甚至上千倍，升高幅度与感染的程度呈正相关。在疾病治愈后其含量急速下降，一周内可恢复正常。临床上CRP一般作为鉴别细菌或病毒感染的一个首选指标，用于自身免疫性及感染性疾病的诊断和监测，以及抗生素疗效观察等。低水平的CRP，又称超敏C-反应蛋白（hs-CRP）。在心血管疾病的诊断中，hs-CRP被认为是心血管炎症病变的生物标志物。在无炎症或感染条件下（代谢稳定下），通常认为：hs-CRP＜1mg/L时为低危险性；1-3mg/L为中度危险；大于3mg/L为高危险性。
　　　PCT是降钙素的前驱物质。正常情况下，降钙素在受到荷尔蒙刺激后仅由甲状腺的C细胞分泌，其浓度通常小于0.1ng/mL。而在促炎症刺激下，特别是在受到细菌感染时，PCT由大量各类细胞产生。当PCT浓度在0.1-0.25ng/mL时，不太可能感染细菌；0.25-0.5ng/mL时可能感染细菌；大于0.5ng/mL时非常可能感染细菌，需要使用抗生素治疗。
　　　PCT与CRP相比，在感染刺激下的3-6小时内即可观察到PCT不断上升，随着感性的加重PCT不断升高。此外，CRP在病毒和细菌性疾病中均能出现，而PCT只有在细菌性感染的疾病中才能出现。因此，PCT作为诊断指标具有快速和特异性强等特点。而联合应用CRP和PCT，将能更为准确的判断炎症的感染程度。在评估心血管疾病的危险程度时，为了避免个体间的差异，hs-CRP的检测通常应排除炎症感染。因此，CRP和PCT的联合将非常重要。因此，PCT和CRP的联合应用，将比二者单独使用时应用范围更广，更便捷，诊断结果也更为准确。
　　　量子点是一种由一定数量的实际原子组成的聚集体，三维尺寸均小于100nm的半导体化合物。它具有发光强度高、激发光谱宽、发射光谱窄、荧光寿命长、表面修饰多功能化和稳定性好等众多优点，在荧光检测领域具有取代传统有机荧光染料的潜力，已成为新一代生物荧光标记物。
发明内容
　　　本发明解决的技术问题是基于CRP与PCT联合诊断的优势，以及量子点的荧光特性，提供一种灵敏度高、快速、准确的PCT/CRP联合诊断试纸。
　　　为解决上述技术问题，本发明采用以下技术方案：
　　　一种CRP/PCT联合诊断试纸，包括设于底板上且依次紧密相连的样品垫、结合垫、反应膜和吸水垫，所述底板设于塑料卡内，所述底板的中部纵向设有一防渗水胶条，所述防渗水胶条将样品垫、结合垫、反应膜和吸水垫分隔为第一样品垫、第二样品垫、第一结合垫、第二结合垫、第一反应膜、第二反应膜、第一吸水垫和第二吸水垫；所述第一结合垫上固定有标记CRP抗体Ⅰ，所述第二结合垫上固定有标记PCT抗体Ⅰ，所述第一反应膜和第二反应膜均设有定量带和质控带。
　　　所述标记CRP抗体Ⅰ为量子点标记的CRP抗体Ⅰ，所述标记PCT抗体Ⅰ为量子点标记的PCT抗体Ⅰ。所述量子点标记的CRP抗体Ⅰ与量子点标记的PCT抗体Ⅰ具有不同的发射波长。
　　　所述第一反应膜上设有第一定量带和第一质控带，所述第一定量带固定有与标记CRP抗体Ⅰ具有不同抗原结合位点的CRP抗体Ⅱ，所述第一质控带固定有标记CRP抗体Ⅰ的二抗。
　　　所述第二反应膜上设有第二定量带和第二质控带，所述第二定量带固定有与标记PCT抗体Ⅰ具有不同抗原结合位点的PCT抗体Ⅱ，所述第一质控带固定有标记PCT抗体Ⅰ的二抗。
　　　所述定量带和质控带的间距为3-5mm。
　　　所述底板为黑色底板。
　　　所述样品垫为玻璃层析膜。
　　　所述试纸具有一个双插槽塑料卡，所述底板设于双插槽塑料卡内。
　　　所述双插擦槽的塑料卡上设有两个并列的加样孔和两个并列的显示窗口。
　　　以上所述CRP/PCT联合诊断试纸的制备方法，包括以下步骤：
　　　(1) 将具有不同发射波长的量子点标记的CRP抗体Ⅰ溶液和量子点标记的PCT抗体Ⅰ溶液分别喷涂于第一结合垫和第二结合垫上并在室温下晾干，于4℃下保存，备用。
　　　所述量子点标记的CRP抗体Ⅰ/PCT抗体Ⅰ溶液为每100mL，1.5-2.5mg/mL的量子点标记的CRP抗体Ⅰ/PCT抗体Ⅰ中分别加入2-5g蔗糖、0.5-1g BSA、0.05-0.1g Tween的溶液。
　　　（2）用标记CRP抗体Ⅰ的二抗溶液在第一反应膜上画线并吹干形成第一质控线，用与标记CRP抗体Ⅰ具有不同抗原结合位点的CRP抗体Ⅱ溶液在第一反应膜上画线并吹干形成第一定量线；用标记PCT抗体Ⅰ的二抗溶液在第二反应膜上画线并吹干形成第二质控线，用与标记PCT抗体Ⅰ具有不同抗原结合位点的PCT抗体Ⅱ溶液在第二反应膜上画线并吹干形成第二定量线；备用。
　　　（3）将第一样品垫、第一结合垫、第一反应膜和第一吸水垫依次固定在防渗水胶条一侧的底板上并使之紧密搭接；将第二样品垫、第二结合垫、第二反应膜和第二吸水垫依次固定在防渗水胶条另一侧的底板上并使之紧密搭接；得CRP/PCT测试条。
　　　所述步骤c所得CRP/PCT测试条插入双插槽塑料卡内。
　　　所述量子点标记的CRP抗体Ⅰ或PCT抗体Ⅰ由以下步骤制备：
　　　（1）清洗量子点：用pH=7.2-7.5的磷酸盐缓冲液清洗量子点至保存量子点的原缓冲液除去，用pH=7.2-7.5的磷酸盐缓冲液保存量子点并超滤浓缩。
　　　所述pH=7.2-7.5的磷酸盐缓冲液的浓度为10mmol/L。
　　　所述超滤浓缩为在20-25℃和5000-7000r/min的条件下，用100K的超滤管离心6-8min。
　　　所述量子点为羧基水溶性量子点；所述羧基水溶性量子点为核壳型量子点或单一化合物形成的量子点。
　　　所述核壳型量子点为ZnS/CdSe或ZnS/CdTe形成的量子点。
　　　所述单一化合物为以下化合物中的任一种：第IIIA族元素和第VA族元素形成的化合物、第IIA族元素和第VIA族元素形成的化合物、第IIB族元素和第VIA族元素形成的化合物、第IVA族元素和第IVA族元素组成的化合物、第IVA族元素和第VIA族元素组成的化合物。具体地，所述单一化合物为GaSb、InAs、InP、InGaAs、InAlAs、MgSe、MgTe、CaS、CaSe、CaTe、SrS、SrSe、SrTe、BaS、BaSe、BaTe、ZnS、ZnSe、ZnTe、CdS、CdSe、CdTe、SiC、SiGe、SiSe、SiTe和SiS中的任一种。
(2)　　　活化：按比例分别称取EDC（1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐）和NHS（N-羟基琥珀酰亚胺），并将EDC和NHS直接加入步骤（1）所得的量子点溶液中，用振荡器混合5-8次后在23-25℃下反应50-70min，得活化量子点；所述EDC、NHS和量子点的摩尔比为（20000-25000）：5000:1。优选地，EDC、NHS和量子点的摩尔比为25000：5000:1。优选地，在0-4℃且空气湿度小于或等于30%的条件下称取EDC和NHS，并将其迅速加入反应液中。更优选地，在0-4℃的冰盒中称取EDC和NHS。
(3)　　　二次清洗量子点：用pH=8.3-8.5的硼酸盐缓冲液清洗步骤（2）所得活化量子点至EDC、NHS和磷酸盐缓冲液除去，用pH=8.3-8.5硼酸盐缓冲液保存活化量子点并超滤浓缩，备用。
　　　所述pH=8.5的硼酸盐缓冲液的浓度为50mmol/L；所述超滤浓缩为在20-25℃和5000-7000r/min的条件下，用100K的超滤管离心6-8min。
　　　（4）清洗抗体：用pH=8.3-8.5的硼酸盐缓冲液清洗CRP抗体Ⅰ或PCT抗体Ⅰ并超滤浓缩除去抗体原保存液中的NaN3，用pH=8.3-8.5硼酸盐缓冲液保存CRP抗体Ⅰ或PCT抗体Ⅰ，备用。
　　　所述pH=8.3-8.5的硼酸盐缓冲液的浓度为50mmol/L；所述超滤浓缩为在1-4℃和3000-5000r/min的条件下，用50K的超滤管离心8-10min。
　　　(5)偶联：将步骤（3）所得活化量子点溶液与步骤（4）所得CRP抗体Ⅰ或PCT抗体Ⅰ溶液混合均匀，在23-25℃及不断摇动下反应5-7h，得CRP抗体Ⅰ或PCT抗体Ⅰ-量子点偶联物反应液；活化量子点与CRP抗体Ⅰ或PCT抗体Ⅰ的摩尔比为1:8-10。优选地，活化量子点与CRP抗体Ⅰ或PCT抗体Ⅰ的摩尔比为1:10，活化量子点的浓度为0.8μmol/L。
　　　(6)分离：通过排阻层析分离步骤（5）的反应液得CRP抗体Ⅰ或PCT抗体Ⅰ-量子点偶联物溶液和CRP抗体Ⅰ或PCT抗体Ⅰ回收液；CRP抗体Ⅰ或PCT抗体Ⅰ-量子点偶联物溶液经超滤浓缩后加入封闭液并在4℃下保存；CRP抗体Ⅰ或PCT抗体Ⅰ回收液经超滤浓缩后回收再利用。排阻层析中使用的排阻层析柱的填料为superdex200或Sephacryl300，流速为1.5ml/min，柱长为40-60cm。所述封闭液为Gly封闭液，Gly的浓度为1mg/mL，溶剂为50mmol/L、pH=8.3-8.5的硼酸盐缓冲液。
　　　向步骤（6）中浓缩后的CRP抗体Ⅰ或PCT抗体Ⅰ-量子点偶联物溶液中加入NaN3，使蛋白-量子点偶联物溶液中NaN3的浓度为0.01-0.05mg/mL，以使蛋白-量子点偶联物溶液可长期保存。
　　　与现有技术相比，本发明的有益效果是：本发明将CRP检测和PCT检测整合于同一检测试纸中，可同时检测CRP和PCT的浓度，不仅提高了检测效率，还同时具备CRP检测和PCT检测的优点。在结合垫上固定量子点标记的抗体，利用量子点优越的荧光特性，提高试纸的灵敏度和准确度。并且本发明制备量子点标记的CRP抗体Ⅰ或PCT抗体Ⅰ的方法，量子点的标记效率高，所制备的标记抗体的稳定性高、活性高，从而进一步提高试纸的灵敏度和准确性。
附图说明
　　　图1为未设双插槽塑料卡的PCT/CRP联合诊断试纸的结构示意图；
　　　图2为设有双插槽塑料卡的PCT/CRP联合诊断试纸的结构示意图；
　　　图3为毛细管电泳鉴定实施例1和实施例2的量子点标记效率；
　　　图4为实施例2制备的PCT-QDs放置不同时间后的荧光光谱图。
具体实施方式
　　　为了更充分理解本发明的技术内容，下面结合具体实施例对本发明的技术方案作进一步介绍和说明。
　　　实施例1
　　　量子点标记的CRP抗体Ⅰ和PCT抗体Ⅰ的制备：
　　　选取发射波长为525nm的羧基水溶性量子点CdTe/Zn进行CRP抗体Ⅰ的标记，记为CdTe/Zn-525；选取发射波长650nm的量子点CdTe/Zn进行PCT抗体Ⅰ的标记，记为CdTe/Zn-650。
　　　取10μL，8μmol/mL的CdTe/Zn-525和10μL，8μmol/mL的CdTe/Zn-650分别置于10μL的EP（eppendorf）管中，并分别加入2mL，10mmol/L，pH=7.2的磷酸盐缓冲液，混合均匀后分别用100K超滤管在5000r/min,25℃下离心6min，分别回收CdTe/Zn-525和CdTe/Zn-650于EP管中。重复以上步骤三次，并最后使量子点溶液的体积不超过50μL。用微量pH计检量子点溶液的pH并调节pH值至7.3。在湿度不超过30%及0-4℃的冰盒中分别称取2mmol EDC和0.5mmol NHS两份并分别迅速加入两种量子点溶液中，涡旋振荡器震荡均匀后分别置摇床上反应60min，反应温度为24℃，分别得活化CdTe/Zn-525和活化CdTe/Zn-650。反应结束后向两活化量子点中分别加入2mL，50mmol/L，pH=8.5的硼酸盐缓冲液，混匀后分别用100K超滤管在5000r/min,25℃条件下离心6min，分别回收活化量子点于EP管中。重复以上步骤三次，最后使两活化量子点溶液的体积分别不超过200μL。用微量pH计检测活化量子点溶液的pH并调节pH值至8.5。
　　　取0.8μmol CRP抗体Ⅰ和0.8μmol PCT抗体Ⅰ分别置于不同EP管中，并分别加入2mL，50mmol/L，pH=8.5硼酸盐缓冲液，混匀后用50K超滤管在3000r/min,4℃下离心10min；重复以上步骤三次，最后使CRP抗体Ⅰ溶液和PCT抗体Ⅰ溶液体积不超过50μL。用微量pH计检测抗体溶液的pH并调节pH值至8.5。
　　　将CdTe/Zn-525溶液与CRP抗体Ⅰ溶液混合，CdTe/Zn-650溶液与PCT抗体Ⅰ溶液混合，混匀后分别在摇床上反应6h，反应温度23-25℃，得CRP抗体Ⅰ-CdTe/Zn-525复合物反应液和PCT抗体Ⅰ-CdTe/Zn-650复合物反应液。用毛细管电泳检测CdTe/Zn-650与PCT抗体Ⅰ的偶联情况，检测结果如图3所示，相对PCT抗体Ⅰ-CdTe/Zn-650复合物的峰面积，量子点（QDs）的峰面积很小，即量子点的标记效率很高。
　　　分别用排阻层析分离两种反应液得CRP抗体Ⅰ-CdTe/Zn-525复合物和PCT抗体Ⅰ-CdTe/Zn-650复合物，并用100K超滤管将复合物浓缩到200μL以内，检测复合物的浓度后加入Gly（甘氨酸）浓度为1mg/mL的Gly封闭液，并置于4℃保存，备用。
　　　排阻层析柱的填料为superdex200，流速为1.5mL/min，凝胶长40cm。
　　　结合垫：
　　　第一结合垫喷涂液为每100mL，1.5-2.5mg/mL的量子点标记的CRP抗体Ⅰ中分别加入2-5g蔗糖、0.5-1g BSA、0.05-0.1g Tween的溶液；第二结合垫喷涂液为每100mL，1.5-2.5mg/mL的量子点标记的PCT抗体Ⅰ中分别加入2-5g蔗糖、0.5-1g BSA、0.05-0.1g Tween的溶液。
　　　将第一结合垫喷涂液均匀喷涂于第一结合垫21上，将第二结合垫喷涂液均匀喷涂于第二结合垫22上，室温晾干，置于4℃保持，备用。
　　　反应膜：
　　　用标记CRP抗体Ⅰ的二抗溶液在第一反应膜上画线并吹干形成第一质控线，用与标记CRP抗体Ⅰ具有不同抗原结合位点的CRP抗体Ⅱ溶液在第一反应膜上画线并吹干形成第一定量线；用标记PCT抗体Ⅰ的二抗溶液在第二反应膜上画线并吹干形成第二质控线，用与标记PCT抗体Ⅰ具有不同抗原结合位点的PCT抗体Ⅱ溶液在第二反应膜上画线并吹干形成第二定量线；备用。
　　　标记CRP抗体Ⅰ的二抗溶液的浓度为1.2mg/ml；CRP抗体Ⅱ溶液的浓度为0.8mg/ml；标记PCT抗体Ⅰ的二抗溶液的浓度为1.2mg/ml；PCT抗体Ⅱ溶液的浓度为1.5mg/ml。
　　　PCT/CRP联合诊断试纸的组装：
　　　参照图1和图2，试纸的底板为黑色底板，并且在底板的中部纵向设置一条防渗水胶条5，防渗水胶条5将底板分为左右两半。第一样品垫11和第二样品垫12为玻璃层析膜。将第一样品垫11、第一结合垫21、第一反应膜31和第一吸水垫41依次设于底板的一半并紧密搭接，将第二样品垫12、第二结合垫22、第二反应膜32和第二吸水垫42依次设于底板的另一半并紧密搭接。将固定了样品垫、结合垫、反应膜和吸水垫的底板插入双插槽塑料卡内。双插槽塑料卡6上设有两个并列的加样孔62和两个并列的显示窗口61。两加样孔62分别位于第一样品垫11和第二样品垫12的上方，通过加样孔62把样品滴至样品垫上。两显示窗口61分别位于第一反应膜31和第二反应膜32的上方，通过显示窗口61分别观察和测量第一质控带312、第二质控带322、第一定量带311和第二定量带321的检测情况。
　　　PCT/CRP联合诊断试纸的使用：
　　　分别向第一反应膜31和第二反应膜32上滴加20μL检测用缓冲液，并层析反应20s。然后将90μL全血或血清样品分别滴加于第一样品垫11和第二样品垫12上，并层析反应8min。将反应后的试纸置于365nm紫外灯下观察反应膜上质控带的情况，若条带清晰则将试纸置于紫外荧光定量仪中获取定量带和质控带的荧光强度，校正后根据已制作的标准曲线进行定量操作。输出检测报告，并根据CRP和PCT的值进行临床综合判断。
　　　所述检测用缓冲液为pH=7.4且含1wt% BSA和0.05% Tween20的PBS缓冲液。
　　　实施例2
　　　本实施例与实施例1的不同之处为：制备量子点标记的PCT抗体Ⅰ的工艺参数不同。
　　　取10μL 8μmol/mL的CdTe/ZnS-650于EP管中，并向EP管中加入2mL 10mmol/L，pH=7.2磷酸盐缓冲液，混匀后用100K超滤管在5000r/min，25℃条件下离心6min，回收量子点于EP管中。重复以上步骤三次，并使最后一次CdTe/ZnS-650溶液的体积不超过50μL。用微量pH计检测量子点溶液的pH并调节pH值至7.2。在湿度不超过30%及0-4℃的冰盒中称取2mmol EDC和0.5mmol NHS并迅速加入CdTe/ZnS-650溶液中，用涡旋振荡器震荡均匀后置于摇床上反应50min，反应温度为23℃，得活化CdTe/ZnS-650溶液。向活化CdTe/ZnS-650溶液中加入2mL 50mmol/L、pH=8.3的硼酸盐缓冲液，混匀后用100K超滤管在5000r/min、20℃条件下离心6min，回收量子点于EP管中。重复以上步骤三次，使最后一次活化CdTe/ZnS-650溶液的体积不超过200μL。用微量pH计检测活化CdTe/ZnS-650溶液的pH并调节pH值至8.3。
　　　取0.8μmol的PCT抗体Ⅰ于EP管中，加入2mL 50mmol/L、pH=8.3硼酸盐缓冲液，混匀后用50K超滤管在3000r/min、1℃条件下离心8min。重复以上步骤三次，使最后一次PCT抗体溶液总体积不超过50μL。用微量pH计检测PCT抗体溶液的pH并调节pH值至8.3。
　　　将活化CdTe/ZnS-650溶液与PCT抗体Ⅰ溶液混合，混匀后置于摇床上反应5h，反应温度23℃，得PCT抗体Ⅰ-CdTe/ZnS-650反应液。用毛细管电泳检测量子点和PCT抗体Ⅰ的偶联情况，如图3所示。
　　　通过排阻层析分离反应液得PCT抗体Ⅰ-CdTe/Zn-650复合物，并用100K超滤管将PCT抗体Ⅰ-CdTe/Zn-650复合物浓缩到200μL以内，测复合物浓度后加入Gly（甘氨酸）浓度为1mg/mL的Gly封闭液，并置于4℃保存，备用。
　　　用荧光分光光度计分别检测本实施例制备的纯化后PCT抗体Ⅰ-CdTe/Zn-650复合物放置5min、1h、24h、3d、10d和30d时的荧光光谱，检测结果如图4所示。图中1为QDs的曲线;2为QDs-PCT，5min的曲线;3为QDs-PCT，1h的曲线;4为QDs-PCT，3h的曲线；5为QDs-PCT，10d的曲线；6为QDs-PCT，30d的曲线。放置不同时间段的PCT-QDs的荧光轻度峰值有降低的趋势，但变化很慢，表明所得PCT-QDs比较稳定。
　　　以上所述仅以实施例来进一步说明本发明的技术内容，以便于读者更容易理解，但不代表本发明的实施方式仅限于此，任何依本发明所做的技术延伸或再创造，均受本发明的保护。

Claims (10)

  1. 一种CRP/PCT联合诊断试纸，包括设于底板上且依次紧密相连的样品垫、结合垫、反应膜和吸水垫，其特征在于，所述底板的中部纵向设有一防渗水胶条，所述防渗水胶条将样品垫、结合垫、反应膜和吸水垫分隔为第一样品垫、第二样品垫、第一结合垫、第二结合垫、第一反应膜、第二反应膜、第一吸水垫和第二吸水垫；所述第一结合垫上固定有标记CRP抗体Ⅰ，所述第二结合垫上固定有标记PCT抗体Ⅰ，所述第一反应膜和第二反应膜均设有定量带和质控带。
  2. 根据权利要求1所述一种CRP/PCT联合诊断试纸，其特征在于，所述标记CRP抗体Ⅰ为量子点标记的CRP抗体Ⅰ，所述标记PCT抗体Ⅰ为量子点标记的PCT抗体Ⅰ；所述量子点标记的CRP抗体Ⅰ与量子点标记的PCT抗体Ⅰ具有不同的发射波长。
  3. 根据权利要求2所述一种CRP/PCT联合诊断试纸，其特征在于，所述第一反应膜上设有第一定量带和第一质控带，所述第一定量带固定有与标记CRP抗体Ⅰ具有不同抗原结合位点的CRP抗体Ⅱ，所述第一质控带固定有标记CRP抗体Ⅰ的二抗。
  4. 根据权利要求3所述一种CRP/PCT联合诊断试纸，其特征在于，所述第二反应膜上设有第二定量带和第二质控带，所述第二定量带固定有与标记PCT抗体Ⅰ具有不同抗原结合位点的PCT抗体Ⅱ，所述第一质控带固定有标记PCT抗体Ⅰ的二抗。
  5. 根据权利要求4所述一种CRP/PCT联合诊断试纸，其特征在于，所述定量带和质控带的间距为3-5mm。
  6. 根据权利要求5所述一种CRP/PCT联合诊断试纸，其特征在于，所述底板为黑色底板。
  7. 根据权利要求6所述一种CRP/PCT联合诊断试纸，其特征在于，所述试纸具有一个双插槽塑料卡，所述底板设于双插槽塑料卡内。
  8. 一种如权利要求1-6任一项所述CRP/PCT联合诊断试纸的制备方法，其特征在于，包括以下步骤：
    a. 　　　将具有不同发射波长的量子点标记的CRP抗体Ⅰ溶液和量子点标记的PCT抗体Ⅰ溶液分别喷涂于第一结合垫和第二结合垫上并在室温下晾干，于4℃下保存，备用；
    　　　所述量子点标记的CRP抗体Ⅰ/PCT抗体Ⅰ溶液为每100mL，1.5-2.5mg/mL的量子点标记的CRP抗体Ⅰ/PCT抗体Ⅰ中分别加入2-5g蔗糖、0.5-1g BSA、0.05-0.1g Tween的溶液；
    b. 用标记CRP抗体Ⅰ的二抗溶液在第一反应膜上画线并吹干形成第一质控线，用与标记CRP抗体Ⅰ具有不同抗原结合位点的CRP抗体Ⅱ溶液在第一反应膜上画线并吹干形成第一定量线；用标记PCT抗体Ⅰ的二抗溶液在第二反应膜上画线并吹干形成第二质控线，用与标记PCT抗体Ⅰ具有不同抗原结合位点的PCT抗体Ⅱ溶液在第二反应膜上画线并吹干形成第二定量线；备用；
    c. 将第一样品垫、第一结合垫、第一反应膜和第一吸水垫依次固定在防渗水胶条一侧的底板上并使之紧密搭接；将第二样品垫、第二结合垫、第二反应膜和第二吸水垫依次固定在防渗水胶条另一侧的底板上并使之紧密搭接；得CRP/PCT测试条。
  9. 根据权利要求8所述CRP/PCT联合诊断试纸的制备方法，其特征在于，将步骤c所得CRP/PCT测试条插入双插槽塑料卡内。
  10. 根据权利要求8所述CRP/PCT联合诊断试纸的制备方法，其特征在于，所述量子点标记的CRP抗体Ⅰ或PCT抗体Ⅰ由以下步骤制备：
    a. 用pH=7.2-7.5的磷酸盐缓冲液清洗羧基水溶性量子点，然后向其中加入EDC和NHS，所述EDC、NHS和量子点的摩尔比为20000-25000：5000:1，混合均匀后在23-25℃下反应50-70min得活化量子点；用pH=8.3-8.5的硼酸盐缓冲液清洗活化量子点至EDC、NHS和磷酸盐缓冲液除去，用pH=8.3-8.5硼酸盐缓冲液保存活化量子点，备用；
    b.将经pH=8.3-8.5的硼酸盐缓冲液清洗后的CRP抗体Ⅰ或PCT抗体Ⅰ与步骤a所得活化量子点溶液混合均匀，所述活化量子点与CRP抗体Ⅰ或PCT抗体Ⅰ的摩尔比为1:8-10，在23-25℃下反应5-7h得反应液；反应液经排阻层析得量子点标记的CRP抗体Ⅰ或PCT抗体Ⅰ溶液。
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111983225A (zh) * 2020-05-07 2020-11-24 中国人民解放军军事科学院军事医学研究院 轮状病毒量子点免疫层析试纸条的制备方法
CN112285364A (zh) * 2020-11-05 2021-01-29 南京申基医药科技有限公司 一种联合检测风湿三项的试剂盒及其制备方法
CN115308406A (zh) * 2022-07-19 2022-11-08 杭州协合医疗用品有限公司 一种双样品垫胶体金免疫层析试纸条

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103487574B (zh) * 2013-10-16 2015-06-17 深圳市金准生物医学工程有限公司 一种量子点标记免疫球蛋白的方法
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WO2016065883A1 (zh) * 2014-10-28 2016-05-06 广州万孚生物技术股份有限公司 Pct与crp定量联检层析试纸条及其制备方法
CN105467116A (zh) * 2015-11-28 2016-04-06 宁波美康生物科技股份有限公司 一种便捷式降钙素原检测试剂盒
CN106526192A (zh) * 2016-09-13 2017-03-22 江苏量点科技有限公司 量子点‑抗体荧光探针的制备方法、探针及试纸条
CN108445210A (zh) * 2018-02-09 2018-08-24 深圳市梓健生物科技有限公司 联合检测人寨卡病毒IgG和IgM抗体的试剂盒及其制备方法

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1811449A (zh) * 2006-02-23 2006-08-02 上海交通大学 量子点标记快速免疫层析试纸条的检测方法
WO2008008349A2 (en) * 2006-07-11 2008-01-17 Drexel University Methods of quantitatively assessing inflammation with biosensing nanoparticles
CN101339196A (zh) * 2008-07-29 2009-01-07 上海师范大学 量子点标记免疫层析试纸快速检测膀胱癌方法
CN102520173A (zh) * 2011-12-29 2012-06-27 深圳康美生物科技股份有限公司 一种定量检测肌酸激酶同工酶的荧光免疫层析方法及其试剂盒
CN102539785A (zh) * 2011-12-29 2012-07-04 深圳康美生物科技股份有限公司 一种全程定量检测c-反应蛋白的荧光免疫层析方法及其试剂盒
CN202583204U (zh) * 2012-02-24 2012-12-05 杭州隆基生物技术有限公司 一种梅毒螺旋体重组抗原检测卡
CN103018456A (zh) * 2012-11-29 2013-04-03 深圳市伯劳特生物制品有限公司 降钙素原检测试纸条及其制备方法
CN203519616U (zh) * 2013-10-16 2014-04-02 深圳市大爱医疗科技有限公司 一种crp/pct联合诊断试纸

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN2486973Y (zh) * 2001-06-20 2002-04-17 陈岩松 多项联合快速检测试剂盒
CN2528017Y (zh) * 2001-09-11 2002-12-25 上海生物芯片有限公司 蛋白质检测芯片
CN101086497A (zh) * 2006-06-09 2007-12-12 杨致亭 一种torch五项联合检测卡
CN101308132A (zh) * 2007-05-17 2008-11-19 上海凯创生物技术有限公司 多联检测试剂盒
CN201522494U (zh) * 2009-04-03 2010-07-07 天津中新科炬生物制药有限公司 双联法便潜血快速诊断层析试纸
CN202351242U (zh) * 2011-11-09 2012-07-25 北京乐普医疗科技有限责任公司 多联试纸免疫层析检测卡
CN102707071A (zh) * 2012-06-26 2012-10-03 南京基蛋生物科技有限公司 Pct/crp联合检测用胶体金试纸条及其制备方法

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1811449A (zh) * 2006-02-23 2006-08-02 上海交通大学 量子点标记快速免疫层析试纸条的检测方法
WO2008008349A2 (en) * 2006-07-11 2008-01-17 Drexel University Methods of quantitatively assessing inflammation with biosensing nanoparticles
CN101339196A (zh) * 2008-07-29 2009-01-07 上海师范大学 量子点标记免疫层析试纸快速检测膀胱癌方法
CN102520173A (zh) * 2011-12-29 2012-06-27 深圳康美生物科技股份有限公司 一种定量检测肌酸激酶同工酶的荧光免疫层析方法及其试剂盒
CN102539785A (zh) * 2011-12-29 2012-07-04 深圳康美生物科技股份有限公司 一种全程定量检测c-反应蛋白的荧光免疫层析方法及其试剂盒
CN202583204U (zh) * 2012-02-24 2012-12-05 杭州隆基生物技术有限公司 一种梅毒螺旋体重组抗原检测卡
CN103018456A (zh) * 2012-11-29 2013-04-03 深圳市伯劳特生物制品有限公司 降钙素原检测试纸条及其制备方法
CN203519616U (zh) * 2013-10-16 2014-04-02 深圳市大爱医疗科技有限公司 一种crp/pct联合诊断试纸

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111983225A (zh) * 2020-05-07 2020-11-24 中国人民解放军军事科学院军事医学研究院 轮状病毒量子点免疫层析试纸条的制备方法
CN112285364A (zh) * 2020-11-05 2021-01-29 南京申基医药科技有限公司 一种联合检测风湿三项的试剂盒及其制备方法
CN115308406A (zh) * 2022-07-19 2022-11-08 杭州协合医疗用品有限公司 一种双样品垫胶体金免疫层析试纸条

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