WO2019029083A1 - 一种趋化素及其衍生多肽的检测测试条及其制备方法 - Google Patents

一种趋化素及其衍生多肽的检测测试条及其制备方法 Download PDF

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WO2019029083A1
WO2019029083A1 PCT/CN2017/116588 CN2017116588W WO2019029083A1 WO 2019029083 A1 WO2019029083 A1 WO 2019029083A1 CN 2017116588 W CN2017116588 W CN 2017116588W WO 2019029083 A1 WO2019029083 A1 WO 2019029083A1
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detection
antibody
zone
test strip
sample
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张键
李梦霞
陈杰
肖天霞
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Shenzhen Institute of Advanced Technology of CAS
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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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

Definitions

  • the invention relates to the field of medical examination, in particular to a test strip for chemokine and a preparation method thereof.
  • Chemotin chemerin is a newly discovered adipokines. It is mainly secreted by adipose tissue in the form of autocrine or paracrine, mainly expressed in white adipose tissue, liver and lung. It is also expressed in adrenal gland, pancreas, placenta, ovary, skin, etc. . Chemerin can bind to its receptors, recruit inflammatory cells to participate in the inflammatory response, regulate insulin sensitivity and adipocyte differentiation, thereby affecting glucose and lipid metabolism, and plays an important role in obesity and metabolic syndrome. In addition, chemerin has been reported to have an inhibitory effect on gonadotropin secretion.
  • chemerin may also be involved in steroid synthesis and hormone secretion in reproductive diseases (gestational diabetes, polycystic ovary syndrome, preeclampsia, etc.) associated with obesity and metabolic abnormalities. It plays an important regulatory role in the process and may become a new target for the prevention and treatment of related diseases.
  • Serum chemerin levels ranged from 300 ⁇ 16 ng/ml to 353.8 ⁇ 18.0 ng/ml, whereas serum chemerin levels in patients with fatty liver were as high as 674.7 ng/ml (Sell H et al, 2010).
  • Eclampsia is a fetal disease, but the mechanism by which the placenta specifically induces the pathogenesis of PE is unclear.
  • the placenta plays a dominant role in the pathogenesis of PE: (1) in the pregnancy of the mole, no eclampsia can occur in the absence of the fetus; (2) In the case of ectopic eclampsia, the symptoms of eclampsia are still present, only the placenta is removed. The symptoms will then disappear; (3) Postpartum eclampsia is caused by a placenta that remains in the uterus. In addition, delivery of the placenta is the only effective treatment for eclampsia.
  • chemerin is highly expressed in obesity, diabetes, hypertension and metabolic syndrome, and in eclampsia.
  • the content of chemerin in serum of pregnant women with eclampsia was significantly higher than that of normal pregnant women.
  • Obesity is a chronic metabolic disease caused by a variety of factors, which is characterized by an abnormal increase in the body fat percentage in the body's fat cells and an increase in the number of cells, and is characterized by excessive deposition of fat in some areas.
  • Simple obesity patients have a uniform distribution of systemic fat, no endocrine disorders, no metabolic disorders, and their families often have a history of obesity.
  • Obesity is a condition caused by the accumulation of body fat, especially triglycerides.
  • Obesity can be divided into two categories: simple obesity and secondary obesity. Most of the obesity we see in the past belongs to the former, and the proportion of simple obesity is as high as 99%.
  • Non-alcoholic fatty liver disease is a common disease of liver disease caused by a series of non-alcoholic liver damage factors, characterized by hepatic parenchymal cell fat accumulation, which can be derived from simple fatty liver. , through nonalocholic steatohepatitis (NASH), develops into liver fibrosis and even causes liver cirrhosis. End-stage liver disease such as hepatocellular carcinoma (HCC) or liver failure.
  • NAFLD hepatocellular carcinoma
  • NAFLD has become the second largest liver disease in China after viral hepatitis, but the specific pathogenesis has not yet been proven. Therefore, the development of drugs and clinical treatment are blocked.
  • NAFLD Newcastle disease virus
  • cirrhosis rate 15% to 25% within 10 years, of which 30% to 40% will die from liver cancer, liver failure and transplant liver recurrence.
  • NAFLD has become the leading cause of global chronic liver disease and liver enzyme abnormalities. NAFLD is not an incurable disease, but without paying attention to it and taking no reasonable treatment measures, it will affect the prognosis.
  • the development of the disease progresses from simple fatty liver, steatohepatitis, liver fibrosis to cirrhosis. Hepatocarcinoma occurs in patients with cirrhosis and has been recognized by medicine.
  • adipokines such as adiponectin, leptin, and resistin are involved in the development of insulin resistance (IR) and NAFLD.
  • Chemerin is a newly identified adipokines in 2007. Chemerin is also known as tazarotene and to gene 2 or retinoic acid receptor-reactive protein 2.
  • One of the receptors for chemerin is called chemokine receptor-like 1, CMKLR1, or ChemR23 and DEZ.
  • chemerin has two other receptors, CCRL2 and GPR1.
  • chemerin binds to three receptors, but the effects are quite different. It has recently been found that the level of chemerin in patients with NAFLD is higher than that in normal subjects. The expression of hepatic chemerin and its receptor CMKLR1 is altered in NAFLD, suggesting that chemerin and its receptor may be involved in the pathogenesis of NAFLD.
  • PCOS Polycystic ovary syndrome
  • the incidence rate is 6-10% of women of childbearing age. It is mainly characterized by menstrual thinning or amenorrhea, infertility, ovarian polycystic Sexual change, obesity, hairy, hyperandrogenism, etc. It is a syndrome caused by the imbalance of the hypothalamic-pituitary-ovarian axis, and it is also a metabolic disorder. This dysfunctional disease of reproductive function and metabolic function is usually accompanied by other diseases such as type 2 diabetes, endometrial cancer and obesity (Dunaif, 1997).
  • PCOS cardiovascular disease 2019
  • drug treatment of PCOS has replaced surgical treatment as a first-line treatment.
  • Chemerin is a newly discovered adipokines associated with energy metabolism, obesity, immune and reproductive function regulation (Iannone and Lapadula, 2011).
  • serum adipokines in women with normal women and women with polycystic ovary syndrome found that women with polycystic ovary syndrome had higher concentrations of chemerin in their serum and their content increased with increasing insulin concentration (Bozaoglu et al) ., 2009; Tan et al,, 2009).
  • the Elisa board is generally a 96-well plate. The whole plate is expensive. It is economical to wait for a sample of multiple patients to be tested together. It is not suitable for the detection of single or several patient samples, and the time waiting for multiple patients is uncertain.
  • the invention utilizes the immunocolloidal gold technology and the chromatographic principle to design, and utilizes the principle of the double-anti-sandwich method to design a test strip to detect the concentration of the chemotactic chemerin protein in the sample.
  • test strip for detecting a chemokine which comprises a detection membrane for detecting a membrane a binding zone containing a first antibody labeled with a label is disposed upstream, and a sampling zone is disposed upstream of the binding zone, wherein a detection zone and a control zone are disposed on the detection membrane, and the control zone is located downstream of the detection zone; wherein the detection zone
  • the second antibody is coated with a second antibody, and the control region is coated with a secondary antibody against the first antibody; both the first antibody and the second antibody are capable of specifically binding to the chemokine.
  • the binding region comprises a first antibody labeled with a label selected from the group consisting of colloidal gold, colloidal silver, colloidal black or dyed latex particles, preferably colloidal gold.
  • the first antibody contained in the binding region is selected from an antibody of a chemotactic functional fragment, preferably an antibody of a C-23 polypeptide, wherein the sequence of the C-23 polypeptide is:
  • the second antibody is selected from the group consisting of a chemotactic antibody.
  • a chemokine monoclonal antibody is preferred.
  • an absorption pad is disposed downstream of the detection film.
  • the first antibody labeled with the label in the binding region is used in an amount of 0.2 ⁇ g/cm 2 or more, preferably 0.24 ⁇ g/cm 2 or more. That is, the mass of the first antibody labeled with a label per unit area of the binding region.
  • the amount of the second antibody coated in the detection zone is 0.15-5.67 ⁇ g/cm.
  • the detection membrane is selected from the group consisting of Startorius CN95, and the amount of the second antibody coated in the detection zone is 0.15-1.67 ⁇ g/cm, and the detection limit of the sample to be tested is 500- 60ng/ml.
  • the detection membrane is selected from the group consisting of Startorius CN140, and the amount of the second antibody coated in the detection zone is 4.15-5.67 ⁇ g/cm, and the minimum color detection limit of the sample to be tested is respectively 500-60 ng/ml.
  • the detection membrane is selected from Pall Vivid 170, and the amount of the second antibody coated in the detection zone is 1.15-2.67 ⁇ g/cm, and the detection limit of the sample to be tested is 500. -60 ng/ml.
  • the detection membrane is selected from the group consisting of Startorius CN140, and the amount of the second antibody coated in the detection zone is (X ⁇ 15%) ⁇ g/cm, and the detection limit of the minimum coloration is Y ng/ml.
  • the detection membrane is selected from Whatman AE99, and the amount of the second antibody coated in the detection zone is (X ⁇ 15%) ⁇ g/cm, and the detection limit of the minimum color development is Y ng/ml.
  • the amount of the second antibody coated in the detection zone is (X ⁇ 10%) ⁇ g/cm; in a more preferred embodiment, the amount of the second antibody coated in the detection zone is It is (X ⁇ 5%) ⁇ g/cm.
  • the amount of the detection line is selected from any integer of 80-400; in a more preferred embodiment, the amount of the detection line is selected from any integer of 100-300;
  • the amount of the second antibody coated in the detection zone and the minimum color detection limit are shown in Table a.
  • the secondary antibody of the first antibody coated in the control region is used in an amount of 0.8 ⁇ g/cm or more, more preferably 1.0 ⁇ g/cm or more. That is, the single film of the first antibody is used to form a single film when the film is formed. The amount of secondary antibody of the first antibody in the length of the bit.
  • the interval between the detection line and the control line is 3-7 mm, preferably 4-6 mm, more preferably 5 mm.
  • Another aspect of the present invention provides a method for preparing a test strip for detecting a chemokine, comprising the steps of:
  • the sample loading zone, the bonding zone, the detection zone and the absorbent pad are assembled to the bottom plate in sequence.
  • the sample pad treatment liquid is prepared by using an inert protein, a surfactant, a preservative, and a buffer, and the sample application area is immersed in the sample pad treatment liquid, and dried overnight.
  • the sample pad treatment liquid is prepared by using 0.5% ovalbumin, 0.01% teen-20, 0.02% sodium azide preservative, 0.01 M pH 7.0 PBS, and the sample is applied. The area was immersed in the sample pad treatment solution, dried at 37 ° C overnight, and sealed at room temperature.
  • step (2) the first antibody labeled with the label is between pH 8.2 and 8.6 and the final concentration of the first antibody is 40-70 [mu]g/ml.
  • the label-labeled first antibody solution is applied to the binding region in an amount of 0.2 ⁇ g/cm 2 or more.
  • a blocking solution is prepared as an inert protein and a buffer.
  • the second antibody was coated in the detection zone in an amount shown in Table a.
  • the secondary antibody of the first antibody was coated in the control zone in an amount of 1 ⁇ g/cm or more.
  • a blocking solution was prepared with 2% bovine serum albumin, 2% skim milk powder, and 0.01 M, pH 7.4 PBS buffer.
  • test strip for detecting a chemokine in a kit for detecting a chemokine in a sample to be tested.
  • a test strip for detecting a chemokine is prepared for evaluating or diagnosing obesity, fatty liver, nonalcoholic fatty liver disease, liver fibrosis, eclampsia, gestational diabetes, diabetes, and polycystic ovary syndrome. Use in kits for chemokines in kits.
  • a method for detecting a chemokine is provided.
  • the test strip of the present invention is used to add a sample to be tested to a sample application zone, and the result is judged according to the color development of the test strip within 5-15 minutes.
  • the present invention provides a chemokine detecting composition comprising two or more test strips of different minimum chromogenic detection concentrations of the present invention.
  • the method for detecting a chemokine according to the present invention has an ex vivo sample as a test sample.
  • control line is colorless, the test strip is invalid and needs to be re-detected; if there is a red strip on both the control line and the detection line, the result is that the chemerin concentration in the sample exceeds the threshold of the minimum color detection limit D of the test strip, indicating the risk.
  • Early warning if the color of the test line is light, it indicates that the concentration of chemerin in the sample is high, which is a high-risk warning.
  • the concentration of chemerin in the sample is higher, which is an ultra-high risk warning; if the control line has a red bar With the test line being colorless, the result is that the chemerin concentration in the sample is lower than the threshold of the minimum color detection limit D of the test strip, suggesting safety and low risk.
  • the test strip consists of a bottom plate, a nitrocellulose membrane, a gold standard pad containing a gold standard antibody, a sample pad and an absorbent pad.
  • the nitrocellulose membrane is disposed in the middle of the bottom plate, and the gold standard pad and the sample pad containing the gold standard antibody are sequentially attached to one side of the nitrocellulose membrane, and the bottom surface of the nitrocellulose membrane is affixed with water absorption.
  • a test line containing a chemotactic chewingin antibody and a control line containing an anti-mouse antibody IgG.
  • a test strip refers to a device that can be used to detect whether or not a chemokine is contained in a sample to be tested.
  • the test strip includes at least a detection membrane having a detection zone and a control zone.
  • the test strip can be placed in a housing, such as a plastic case, or the test strip can have no outer casing.
  • the detection membrane is a solid support comprising a detection zone and a control zone.
  • the detection membrane can be any solid support, preferably a nitrocellulose membrane.
  • the detection zone, the detection zone, and the detection line are used interchangeably, and both refer to a region on the detection membrane coated with an antibody that binds to a chemokine.
  • control region refers to a region on the detection membrane coated with the secondary antibody of the first antibody attached to the label contained in the gold standard pad.
  • a sample pretreatment membrane is disposed upstream of the binding zone, and the sample pretreatment membrane Ability to treat impurities in the sample that may affect the results of the experiment.
  • the sample pretreatment membrane can be a blood filtration membrane.
  • an antibody has its broadest meaning and includes, for example, but is not limited to, intact antibodies and single chain antibodies, antibody fragments, and chimeric antibodies.
  • the invention combines the theoretical knowledge of medicine, biology and immunology, and develops the concentration of chemerin protein in serum, saliva, urine or tissue fluid by using double-anti-sandwich colloidal gold immunoassay and test strip chromatography principle.
  • the test strip provides new tools for the monitoring and early prevention of metabolic and reproductive diseases in which chemerin secretes abnormalities such as obesity, fatty liver, pre-eclampsia, gestational diabetes and polycystic ovary syndrome.
  • test strip of the invention is convenient and quick to use.
  • red liquid moves to the test area, remove the flat and clean non-adsorbed.
  • the plane of the material starts to count, and the result can be judged according to the color of the test strip within 5-15 minutes.
  • the test strip of the present invention can achieve semi-quantitative. According to the ratio of the amount of the immunogold and the detection line antibody which have been set in Table 2, if the B line of the test strip A is colored, it indicates that the concentration of chemerin in the sample is higher than the corresponding detection limit D, The test strips with different minimum color detection limits can be used to semi-quantitatively measure the range of chemerin content in the sample to be tested.
  • FIG. 1 Western-blot results for both antibodies.
  • Figure 3 Schematic diagram of the test strip structure, where 1, sample pad, 2, gold standard pad, 3, bottom plate, 4, nitrocellulose membrane, 5, detection zone, 6, control zone, 7, absorbent pad.
  • Figure 6 Location, composition and code of each component of the test strip, where A is the control line, anti-mouse antibody IgG, B is the detection line, mouse anti-human chemokine antibody, C is the gold standard pad, mouse The anti-human C-23 antibody is immunogold, and D is the sample to be tested.
  • a KLH-protein conjugate was prepared by coupling a C-23 polypeptide and a recombinant Chemerin protein, respectively, using a ReadiLink KLH Conjugation Kit (AAT BIOQUEST, 5502) kit, and the conjugate was purified.
  • mice 6-8 weeks old female Balb/c mice were immunized with the above KLH-protein conjugate, and after immunization for 4 times, mouse spleen cells were aseptically taken for cell fusion.
  • Enzyme-linked immunosorbent assay The supernatant of the hybridoma cell line positive for positive identification was purified by Protein A affinity chromatography. The collected antibody solution was lyophilized.
  • sample pad 0.5% ovalbumin, 0.01% teen-20, 0.02% sodium azide preservative, 0.01 M pH 7.0 PBS, make sample pad treatment solution, soak the sample pad in the sample After the pad treatment solution, it was dried overnight at 37 ° C, and sealed at room temperature.
  • Immunogold was prepared using Gold-in-a-box Conjugation Kit (20 nm), pH 8.4, concentration of 56 ⁇ g/ml, and the immunogold was diluted to 50 ⁇ g/ml. And coated on a colloidal gold bond pad, the amount is about 0.24 ⁇ g / cm 2 , dried at 37 ° C, and stored at 4 ° C for use.
  • NC solid phase nitrocellulose membrane
  • 2% bovine serum albumin, 2% skim milk powder and 0.01 M, pH 7.4 PBS buffer were mixed, and the resulting blocking solution was filtered through a 0.45 ⁇ m filter.
  • the NC membrane was dried for 1 hour at 37 ° C.
  • the chemoattractant Chemerin recombinant protein was used as a test sample for chromatographic test to observe the coloration and optimize the concentration of spotted protein.
  • the effect on the sample detection line was tested with different NC membranes and adjusting the control line (A), detection line (B), and gold label antibody concentration (C) on the test strip.
  • the nitrocellulose membrane coated with protein On the bottom plate, the nitrocellulose membrane coated with protein, the sample pad made of gold standard pad and glass cellulose film, and the absorbent pad made of absorbent paper are assembled according to Fig. 3, in the bottom plate.
  • a sample pad, a gold standard pad, a nitrocellulose membrane, and an absorbent paper are disposed in sequence, wherein the nitrocellulose membrane is provided with a detection zone and a control zone.
  • the assembled cardboard is cut longitudinally and cut into strips having a width of 3 mm, which is the finished product of the antibody test strip.
  • NC membrane models and control line (A), detection line (B), and gold standard antibody concentration (C) on the detection limit of the sample.
  • the detection limit of the chemokine The detection limit of the chemokine.
  • the minimum coloration detection line is in the range of 100-300 ng/ml, and the amount B of mouse anti-human Chemerin antibody is (X ⁇ 15%) ⁇ g/cm, and the minimum color detection limit D
  • Warning value A red band appears on both the control line (A) and the detection line (B), indicating that the chemerin concentration in the sample is above the detection limit of the test strip, which is a high risk value. As shown in Fig. 5, if the detection line (B) is light in color, it indicates that the concentration of chemerin in the sample is high, indicating a high-risk warning; if the detection line (B) is darker, it indicates that the concentration of chemerin in the sample is higher. The prompt is an ultra high risk warning.
  • the invention can also be combined and used by using a plurality of test strips with different minimum detection lines, for example, using two test strips with different minimum color detection lines in combination, and detecting lines of test strips with a minimum color detection line of 240 ng/ml.
  • B A red band appears, and the detection line (B) of the test strip with a minimum color detection line of 300 ng/ml does not show a red band, which proves that the concentration of the chemokine in the sample to be tested is 240 ng/ml- Between 300 ng/ml, the purpose of semi-quantitative can be further achieved.

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Abstract

一种趋化素及其衍生多肽的检测测试条及其制备方法；测试条包括检测膜，检测膜的上游设置有含有以标记物标记的第一抗体的结合区，结合区上游设置有加样区；检测膜还设置有检测区和控制区，控制区位于检测区下游；检测区包被第二抗体，控制区包被含有抗第一抗体的二抗；第一抗体和第二抗体均能与趋化素特异性结合。

Description

一种趋化素及其衍生多肽的检测测试条及其制备方法 技术领域
本发明涉及医学检验领域，具体涉及一种趋化素的检测测试条及其制备方法。
背景技术
趋化素chemerin是新发现的脂肪因子，它主要以自分泌或旁分泌的形式由脂肪组织分泌，主要表达于白色脂肪组织、肝脏和肺，在肾上腺、胰腺、胎盘、卵巢、皮肤等也有表达。chemerin可通过与其受体结合，募集炎性细胞参与炎症反应，调节胰岛素的敏感性和脂肪细胞分化，从而对糖脂代谢产生影响，在肥胖及代谢综合征中起重要作用。此外，chemerin被报道对性腺激素分泌表现出抑制作用，因此，chemerin也可能在与肥胖和代谢异常相关的生殖疾病(妊娠糖尿病、多囊卵巢综合征、子痫前期等)的类固醇合成和激素分泌过程中发挥重要的调节作用，且有可能成为相关疾病预防和治疗的新靶点。
近年来，快节奏的工作和生活方式，高消费的压力，方方面面都挑战着婚育年龄的女性。因此，女性的健康，尤其是女性的生殖健康是不容忽视的社会问题。大量研究表明，子痫、肥胖、脂肪肝、妊娠糖尿病及多囊卵巢综合征患者血清中chemerin蛋白的含量要明显高于普通人。有报道显示，在对肥胖和代谢综合征患者的研究中发现，非肥胖健康人群血清中的chemerin含量为150±11ng/ml～191±14ng/ml，体质指数(BMI)超过28的肥胖症患者血清chemerin含量为300±16ng/ml～353.8±18.0ng/ml，而脂肪肝患者血清chemerin含量甚至高达674.7ng/ml(Sell H et al，2010)。在对相关生殖疾病患者的研究中发现，健康孕妇血清中的chemerin的含量为152±13ng/ml～199.96±28.05ng/ml(Cetin O et al,2017)，妊娠糖尿病患者血清chemerin含量为230ng/ml±17ng/ml～300ng/ml±15ng/ml(Pfau D et al,2010)；多囊卵巢综合征患者血清中chemerin含量为311.07±141.87ng/ml(Wang L et al,2014)；重度子痫患者血清中的chemerin含量为364.72±20.01ng/ml～394.72±25.01ng/ml，轻度子痫患者血清中的chemerin含量也高达322.11±37.6ng/ml(Cetin O et al,2017)，等等。
子痫(Eclampsia)
子痫是一个胎源性的疾病，但胎盘具体如何诱导PE发病的机制不清楚。胎盘在PE发病中起主导地位表现在：(1)在葡萄胎妊娠中，没有胎儿的存在，仍可发生子痫；(2)宫外孕子痫病例中，取出子痫症状仍然存在，只有取出胎盘之后症状才会消失；(3)产后子痫病例是由于在子宫内残留的胎盘所造成的病症。另外，娩出胎盘是子痫唯一而有效的治疗方式。虽然病例机制不清，但子痫患者胎盘有字样细胞侵入浅表、螺旋动脉重塑不完全和螺旋动脉的“急性动脉粥样硬化”的典型病例变化。研究表明脂肪因子chemerin在肥胖、糖尿病、高血压和代谢综合征中和子痫中均高表达。chemerin在子痫孕妇血清中的含量比正常孕妇明显升高。上述研究表明chemerin的高表达和受体CMKLR1通路可能为上述各病症共有的发病因素。另有研究表明，chemerin与肿瘤的恶性程度相关。
肥胖、脂肪肝(fatty liver)
肥胖症是一种由多种因素引起的慢性代谢性疾病，以体内脂肪细胞的体积和细胞数增加致体脂占体重的百分比异常增高并在某些局部过多沉积脂肪为特点。单纯性肥胖患者全身脂肪分布比较均匀，没有内分泌紊乱现象，也无代谢障碍性疾病，其家族往往有肥胖病史。
肥胖是体内脂肪，尤其是甘油三酯积聚过多而导致的一种状态。肥胖可分为单纯性肥胖和继发性肥胖两大类。平时我们所见到的肥胖多属于前者，单纯性肥胖所占比例高达99％。
肥胖者的死亡率比正常体重者有明显的增高，随着体重的增加，死亡率也有所增加。研究表明，肥胖者因糖尿病而死亡者比正常体重组明显增高为38.3％(男性)及37.2％(女性)；其次是肝硬化、阑尾炎、胆石症患者的死亡率，肥胖者也增加一倍左右；心血管、肾病及意外事故的死亡率也较高。
非酒精性脂肪肝病(Non-alcoholic fatty liver disease NAFLD)是目前常见的肝脏疾病是一系列非酒精损肝因素所致的、以肝实质细胞脂肪堆积为主要特征的疾病，可从单纯性脂肪肝，经非酒精性脂肪肝炎(nonalocholic steatohepatitis，NASH)，发展为肝纤维化，甚至导致肝硬化(liver cirrhosis)。肝细胞癌(hepatocellular carcinoma，HCC)或肝功能衰竭等终末期肝病。近年来，随着生活水平的提高及饮食结构的改变，我国的NAFLD呈升高的趋势，NAFLD在我国已成为仅次于病毒性肝炎的第二大肝病，但具体的发病机制还未探明，从而使药物的开发及临床治疗受阻。流行病学调查，成人脂肪肝发病率17％～33％， 其中1/3～1/2为NAFLD，后者10年内肝硬化发生率为15％～25％，其中30％～40％将会死于肝癌、肝衰和移植肝复发。NAFLD已成为全球性慢性肝病和肝酶异常的首要病因。NAFLD并非不治之症，但不加以重视、未采取合理的治疗措施，将会影响预后，其病变的发展过程从单纯性脂肪肝、脂肪性肝炎、肝纤维化发展为肝硬化。而肝癌多发生于肝硬化患者，目前已被医学公认。所以应遵循继续深入探索NAFLD的发病机制、早期诊断、早期干预、长期坚持、整体治疗与个体化用药相结合的方案。目前已有文献报道，多种脂肪因子(Adipokine)如：脂联素(adiponectin)、瘦素(leptin)、抵抗素(resistin)与胰岛素抵抗(Insulin Resistance，IR)和NAFLD的发生发展有关。chemerin是2007年新确认的脂肪因子。chemerin也称作他扎罗汀又到基因2或者视黄酸受体反应蛋白2。chemerin的受体之一，称作类趋化因子受体-1(chemokine receptor-like1,CMKLR1)，或者ChemR23和DEZ。除了CMKLR1，chemerin还有其他两种受体，分别是CCRL2和GPR1。研究显示，chemerin与三种受体结合，但作用很不相同。新近发现，NAFLD患者的chemerin水平比正常人的水平高，NAFLD中肝脏chemerin及其受体CMKLR1的表达有变化，提示：chemerin及其受体可能参与NAFLD的发病。
妊娠糖尿病
孕妇在妊娠期出现或首次被检查到出现糖耐量受损，即被确诊为妊娠糖期尿病(GDM)。妊娠糖期尿病是孕妇在妊娠期常见的一种病，妊娠糖尿病常伴随的并发症包括新生婴儿超重或婴儿脏器过大，围产儿死亡率上升，并提高了妊娠糖尿病患者及后代患二型糖尿病的风险。根据国际糖尿病联盟(IDF)统计，2013年妊娠糖期尿病的发病率达到16.8％，超过了2002年统计的发病率10％左右。妊娠糖期尿病发病率的快速上升，及带来的许多的严峻问题，对于研究妊娠糖期尿病发病机制，寻找治疗靶点非常迫切。
GDM患者糖代谢多数于产后能恢复正常，但将来患II型糖尿病机会增加。糖尿病孕妇的临床经过复杂，母子都有风险，应该给予重视。GDM患者体内chemerin水平明显上升，与IR和胰岛β细胞功能存在一定的相关性(妊娠糖尿病患者血清FABP4、chemerin、Nesfatin-1与胰岛素抵抗和胰岛β细胞功能的关系分析)。
多囊卵巢综合征
多囊卵巢综合征(polycystic ovary syndrome，PCOS)是育龄妇女常见的内分泌紊乱行疾病，发病率占育龄期妇女的6～10％，其主要表现为月经稀发或闭经、不孕、卵巢多囊性变、肥胖、多毛、高雄激素血症等。它既是一组因下丘脑-垂体-卵巢轴的平衡失调引起的综合征，也是一种代谢紊乱行疾病。这种生殖功能和代谢机能的系统失调疾病，通常还伴随着二型糖尿病，内膜癌和肥胖等其他疾病(Dunaif，1997)。在PCOS中高度表达的雄激素水平，也会刺激体内累计的过多脂肪组织，引起代谢性疾病如肥胖、胰岛素抵抗和相关的高胰岛素血症等病症。目前PCOS的药物治疗已取代手术治疗作为一线治疗方法。chemerin就是一个新发现的与能量代谢，肥胖，免疫及生殖功能调控功能相关的脂肪因子(Iannone and Lapadula，2011)。2008年在对于正常妇女和多囊卵巢综合征妇女血清中脂肪因子的比较中发现，多囊卵巢综合征妇女的血清中chemerin的浓度较高，并且其含量随insulin浓度增加而增加(Bozaoglu et al.,2009；Tan et al,，2009)。
现如今检测人体血液与尿液中的chemerin含量方法只有酶联免疫吸附测定。该方法较繁琐，用时较长。
现在的测定人体血液或尿液中趋化素Chemerin含量的方法主要使用人Chemerin酶联免疫吸附测定(ELISA)法进行测定，其缺点也是显而易见的：
1)步骤较繁琐。要按照操作步骤进行，一般需要几个小时至一天左右。
2)耗时。Elisa板一般是96孔板，整板的价格昂贵，要等凑够多个病人的样本一起整板检测比较经济，不适合单个或几个病人样本的检测，等待多个病人的时间不确定；
3)不方便。要通过特定仪器读取信号，只能在有条件的场所，如医院和社康进行检测。
4)昂贵。ELISA板的成本高，需要低温储存环境，需要借助仪器读取结果。
发明内容
为了克服上述问题，亟需对趋化素的检测方法进行改进和突破。本发明利用免疫胶体金技术以及层析原理进行设计，利用了双抗夹心法的原理，设计测试条来检测样品中趋化素chemerin蛋白的浓度。
本发明一个方面提供了一种检测趋化素的测试条，其包括检测膜，检测膜的 上游设置有含有以标记物标记的第一抗体的结合区，结合区上游设置有加样区，其中，检测膜上设置有检测区和控制区，控制区位于检测区下游；其中所述检测区上包被有第二抗体，所述控制区包被有抗第一抗体的二抗；所述第一抗体和第二抗体均能与趋化素特异性结合。
在本发明的技术方案中，结合区中包含以标记物标记的第一抗体，所述标记物选自胶体金、胶体银、胶体黑或染色的乳胶颗粒，优选为胶体金。
在本发明的技术方案中，结合区中包含的第一抗体选自趋化素功能性片段的抗体，优选为C-23多肽的抗体，其中C-23多肽的序列为:
Figure PCTCN2017116588-appb-000001
在本发明的技术方案中，所述第二抗体选自趋化素抗体。优选为趋化素单克隆抗体。
在本发明的技术方案中，所述抗第一抗体的二抗选自抗小鼠抗体IgG。
在本发明的技术方案中，所述检测膜的下游设置有吸水垫。
在本发明的技术方案中，所述检测膜选自硝酸纤维素膜，优选为粒径为5-20μm，爬速为80-250s/4cm；更优选为6-15μm，爬速为80-225s/4cm；最优选为①Sartorius CN95，粒径为15μm，爬速约为90-135s/4cm②Sartorius CN140，粒径为8μm，爬速约为110-165s/4cm③Pall Vivid 170，粒径为6μm，爬速为150-225s/4cm④whatman AE99，粒径为8μm，爬速约为120-160s/4cm。
在本发明的技术方案中，所述结合区的以标记物标记的第一抗体的用量为0.2μg/cm2以上，优选为0.24μg/cm2以上。即结合区单位面积上以标记物标记的第一抗体的质量。
在本发明的技术方案中，所述检测区包被的第二抗体的用量为0.15-5.67μg/cm。
在本发明的一个技术方案中，所述检测膜选自Startorius CN95，所述检测区包被的第二抗体的用量为0.15-1.67μg/cm，待测样品最低显色检测限分别为500-60ng/ml。
在本发明的一个技术方案中，所述检测膜选自Startorius CN140，所述检测区包被的第二抗体的用量为4.15-5.67μg/cm，待测样品最低显色检测限分别为 500-60ng/ml。
在本发明的一个技术方案中，所述检测膜选自Pall Vivid 170，所述检测区包被的第二抗体的用量为1.15-2.67μg/cm，待测样品最低显色检测限分别为500-60ng/ml。
在本发明的一个技术方案中，所述检测膜选自Whatman AE99，所述检测区包被的第二抗体的用量为1.15-2.67μg/cm，待测样品最低显色检测限分别为500-60ng/ml。在本发明的技术方案中，所述检测膜选自Startorius CN95，所述检测区包被的第二抗体的用量为(X±15％)μg/cm，最低显色检测限为Y ng/ml，检测区包被的第二抗体的用量与最低显色检测限关系为Y＝-322.23X+360.79，最低显色检测线Y选自60-500的任意整数。
在本发明的技术方案中，所述检测膜选自Startorius CN140，所述检测区包被的第二抗体的用量为(X±15％)μg/cm，最低显色检测限为Y ng/ml，检测区包被的第二抗体的用量与最低显色检测限关系为Y＝-317.51X+1628.6，最低显色检测线Y选自60-500的任意整数。
在本发明的技术方案中，所述检测膜选自Pall Vivid 170，所述检测区包被的第二抗体的用量为(X±15％)μg/cm，最低显色检测限为Y ng/ml，检测区包被的第二抗体的用量与最低显色检测限关系为Y＝-309.64X+665.17；最低显色检测线Y选自60-500的任意整数。
在本发明的技术方案中，所述检测膜选自Whatman AE99，所述检测区包被的第二抗体的用量为(X±15％)μg/cm，最低显色检测限为Y ng/ml，检测区包被的第二抗体的用量与最低显色检测限用量关系为Y＝-316.91X+668.99，最低显色检测线Y选自60-500的任意整数。
在优选的技术方案中，所述检测区包被的第二抗体的用量为(X±10％)μg/cm；在更优选的技术方案中，所述检测区包被的第二抗体的用量为(X±5％)μg/cm。
在优选的技术方案中，所述检测线用量选自80-400的任意整数；在更优选的技术方案中，所述检测线用量选自100-300的任意整数；
在本发明的技术方案中，所述检测区包被的第二抗体的用量与最低显色检测限如表a所示。
表2a
Figure PCTCN2017116588-appb-000002
在本发明的技术方案中，所述控制区包被的第一抗体的二抗的用量为0.8μg/cm以上，更优选为1.0μg/cm以上。即以第一抗体的二抗进行划膜形成时划膜的单 位长度上第一抗体的二抗的用量。
在本发明的技术方案中，所述检测线与控制线之间的间隔为3-7mm，优选为4-6mm，更优选为5mm。
本发明另一个方面提供了一种检测趋化素的测试条的制备方法，其包括如下步骤：
(1)制备加样区，以样品垫处理液处理加样区；
(2)制备结合区：以标记物标记的第一抗体，并将其涂布在结合区；
(3)制备检测膜：稀释第二抗体及第一抗体的二抗，分别包被在检测膜上，形成检测区和控制区，干燥后以封闭液进行封闭；
(4)组装测试条：依次将加样区、结合区、检测区和吸水垫组装至底板上。在步骤(1)中，以惰性蛋白、表面活性剂、防腐剂以及缓冲液制备样品垫处理液，将加样区浸泡于样品垫处理液后，干燥过夜。
进一步地，在步骤(1)中，以0.5％卵清蛋白，0.01％teen-20，0.02％叠氮化钠防腐剂，0.01M pH7.0的PBS，制成样品垫处理液，将加样区浸泡于样品垫处理液后，37℃干燥过夜，室温密封保存。
在步骤(2)中，以标记物标记的第一抗体在pH在8.2-8.6之间，第一抗体最终浓度为40-70μg/ml。
进一步地，在步骤(2)中，标记物标记的第一抗体溶液涂布在结合区的用量为0.2μg/cm2以上。
在步骤(3)中，以惰性蛋白和缓冲液制备封闭液。并以表a所示用量将第二抗体包被在检测区。以1μg/cm以上的用量将第一抗体的二抗包被在控制区。
进一步地，在步骤(3)中，以2％的牛血清蛋白、2％脱脂奶粉和0.01M、pH7.4的PBS缓冲液制备封闭液。
本发明再一个方面提供了一种检测趋化素的测试条在制备检测待测样品中趋化素的试剂盒中的用途。
本发明再一个方面提供了一种检测趋化素的测试条在制备评估或诊断肥胖、脂肪肝、非酒精性脂肪肝病、肝纤维化、子痫、妊娠糖尿病、糖尿病及多囊卵巢综合征的试剂盒中趋化素的试剂盒中的用途。
本发明再一个方面提供了一种检测趋化素的方法，采用本发明的测试条，将待测样品加到加样区，5-15分钟内根据测试条的显色判断结果。
本发明再一个方面提供了一种趋化素检测组合物，其中包含2条以上本发明的不同最低显色检测浓度的测试条。
本发明所述的检测趋化素的方法已离体样品为检测样品。
本发明所述的检测趋化素的方法可以为诊断和治疗目的，或非诊断和治疗目的进行检测。
如果控制线无色，则测试条失效，需重新检测；如果控制线与检测线均出现红色条带，则结果为样品中chemerin浓度超过该试纸条最低显色检测限D的阈值，提示风险预警，若检测线颜色较浅，则提示样品中chemerin浓度高，为高风险预警，若检测线颜色较深，则提示样品中chemerin浓度较高，为超高风险预警；若控制线出现红色条带，检测线无色，则结果为样品中chemerin浓度低于该试纸条最低显色检测限D的阈值，提示安全、低风险。
所述检测测试条由底板、硝酸纤维素膜、含有金标抗体的金标垫、样品垫和吸水垫组成。所述的硝酸纤维素膜设置在底板的中部，硝酸纤维素膜的一侧底板上依次贴有含金标抗体的金标垫和样品垫，硝酸纤维素膜另一侧的底板上贴有吸水垫，所述硝酸纤维素膜中部有一条含有趋化素Chemerin抗体的检测线和一条含有抗小鼠抗体IgG的控制线。
在本发明中待测样品是指将要分析其是否包含趋化素的任何材料。待测样品的形式优选为液体形式，代表性的样品包括单不限于体液，例如，全血、血浆、血清、唾液、组织液或尿液。待测样品也可以是固体，但应在测试前溶解或者提取。
在本发明中，测试条是指能够用来检测待测样品中是否含有趋化素的装置。测试条至少包括具有检测区和控制区的检测膜。测试条可以放置在外壳，例如塑料壳中，或者，测试条也可以没有外壳。
在本发明中，检测膜是一种固体支持物，其包括检测区和控制区。检测膜可以使任何固体支持物，优选为硝酸纤维素膜。
在本发明中，检测区、检测带和检测线可以互换使用，都是指检测膜上包被有结合趋化素的抗体的区域。
在本发明中，控制区、控制带、控制线或控制线可以互换使用，是指检测膜上包被有金标垫中包含的以标记物附着的第一抗体的二抗的区域。
在本发明中，任选地，结合区上游设置有样品预处理膜，所述样品预处理膜 能够对待样品中的可能影响实验结果的杂质。样品预处理膜可以为滤血膜。
在本发明中，抗体为其最宽含义，包括例如，但不限于完整抗体及单链抗体、抗体片段和嵌合抗体。
本发明结合医学、生物学和免疫学理论知识，利用双抗夹心法胶体金免疫技术及测试条层析原理，开发出能够检测血清、唾液、尿液或组织液中趋化素(chemerin)蛋白浓度的测试条，为肥胖、脂肪肝、先兆子痫、妊娠糖尿病及多囊卵巢综合征等chemerin分泌异常的代谢和生殖疾病的监测和早期预防提供新的工具。
有益效果
1、本发明的测试条使用方便、快捷。使用时仅需将测试条有箭头标志的一段垂直浸入装有样品的溶液中纸质箭头下端标记横线处，至少3秒，当红色液体移行至测试区时，取出平放与干净的非吸附材料的平面，开始计时，5-15分钟内根据试条的显色即可判断结果
2、本发明的测试条可以实现半定量。通过表2中已经设定的免疫金与检测线抗体的量的配比，若测试条A线B线均显色，则提示样品中chemerin的浓度为高于对应检测限D以上的风险值，利用不同最低显色检测限的试纸条，可以半定量测出被检样品中chemerin的含量范围。
3、本发明的测试条经济，不需要借助额外仪器，仅根据显色判定结果；常温保存，可以家用。
附图说明
图1：两种抗体Dot-blot结果。
图2：两种抗体Western-blot结果。
图3：测试条结构示意图，其中1、样品垫，2、金标垫，3、底板，4、硝酸纤维素膜，5、检测区，6、控制区，7、吸水垫。
图4：使用方法示意图，其中样品浸入深度不能超过标记横线。
图5：检测结果说明图。
图6：测试条各组分的位置、成分及代号，其中，A为控制线，抗小鼠抗体IgG，B为检测线，小鼠抗人趋化素抗体，C为金标垫，小鼠抗人C-23抗体免疫金，D为待测样品。
具体实施方式
下面结合具体的实施例来进一步阐述本发明。应理解，这些实施例仅用于说明本发明，而不是用来限制本发明的范围。下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
主要实验材料：样品垫、硝酸纤维素(NC)膜、胶体金垫、吸水纸、底板均购自上海杰一生物科技有限公司；C-23多肽为Chemerin蛋白中的一段功能性序列(CLRVQRAGEDPHSFYFPGQFAFS SEQ ID No.1)，由深圳先进技术研究院生殖健康研究室发现，并由强耀生物科技有限公司合成，重组Chemerin蛋白由深研科技有限公司合成，金标试剂盒购自欣博盛生物科技有限公司。本发明所用试验材料均可通过市售途径获得。
实施例1C-23抗体及重组Chemerin蛋白抗体的制备：
(1)使用ReadiLink KLH Conjugation Kit(AAT BIOQUEST，5502)试剂盒分别偶联C-23多肽及重组Chemerin蛋白，制备KLH-蛋白偶联物，并纯化该偶联物。
(2)分别使用上述KLH-蛋白偶联物免疫6-8周龄雌性Balb/c小鼠，免疫4次后，无菌取小鼠脾脏细胞进行细胞融合。
(3)细胞融合前2周复苏骨髓瘤细胞，前1-2天制备并培养小鼠腹腔巨噬细胞，将所得脾细胞与骨髓细胞共培养于含腹腔巨噬细胞的HAT培养基内培养，经常观察杂交细胞瘤情况，待其长至孔底面积1/10以上时吸出上清供抗体检测。
(4)酶联免疫吸附剂测定方法检测所得两种抗体的效价。
(5)酶联免疫吸附剂测定方法鉴定阳性的杂交瘤细胞株上清用Protein A亲和层析法纯化抗体。将收集的抗体溶液冻干。
(6)使用免疫斑点法及蛋白质印迹法验证两种抗体。结果见附图1、附图2。
实施例2胶体金标记的C-23抗体的制备：
使用Gold-in-a-box Conjugation Kit金标试剂盒制备胶体金
(1)将金溶胶涡旋重悬，将0.5ml裸金溶胶放在10个干净的Ep管中；
(2)将pH1-10的标签贴在Ep管上，使用pH值的图表(表1)，在每个试管中添加不同量试剂盒中所配的缓冲液，涡旋混匀；
(3)将每个Ep管低速涡旋，加入C-23抗体溶液14μl(2.5mg/ml)，室温放置30min使反应完全；
(4)加Blocking Stabilizer Soltion 50μl停止反应，在室温下终止反应16h。
(5)为了有效地干燥胶体金，添加0.05ml干燥缓冲液，彻底混合。涂布在胶体金膜上，将其放在37℃孵化一晚。
(6)经检验，pH＝8.4的胶体金较稳定，所得最终抗体浓度为56μg/ml。
表1 胶体金试剂盒pH图表
EP管编号 pH 缓冲液A 缓冲液B
1 5.4 9μl 1μl
2 6.6 8μl 2μl
3 7.3 6μl 4μl
4 7.8 4μl 6μl
5 8.2 2μl 8μl
EP管编号 pH 缓冲液C 缓冲液D
6 8.4 10μl 0μl
7 8.8 8μl 2μl
8 9.2 6μl 4μl
9 9.6 4μl 6μl
10 10.1 2μl 8μl
实施例3制作过程
(1)样品垫的制备：0.5％卵清蛋白，0.01％teen-20，0.02％叠氮化钠防腐剂，0.01M pH7.0的PBS，制成样品垫处理液，将样品垫浸泡于样品垫处理液后，37℃干燥过夜，室温密封保存。
(2)金标垫的制备：使用Gold-in-a-box Conjugation Kit金标试剂盒(20nm)制备免疫金，pH值为8.4，浓度为56μg/ml，稀释该免疫金到50μg/ml，并涂布于胶体金结合垫上，用量约为0.24μg/cm2，37℃干燥，4℃保存备用。
(3)固相硝酸纤维素膜(NC)的制备：2％的牛血清蛋白、2％脱脂奶粉和0.01M、pH7.4的PBS缓冲液混合后用0.45μm滤膜过滤得到的封闭液。用pH＝7.4的PBS缓冲液稀释小鼠抗人Chemerin抗体及抗小鼠二抗，以不同的蛋白浓度点样于硝酸纤维素膜上作为检测线和控制线，室温干燥后用封闭液封闭 NC膜1小时，37℃干燥备用。以趋化素Chemerin重组蛋白为检测样品进行层析试验，观察显色情况，优化点样蛋白浓度。以不同的NC膜，并调节测试条上的控制线(A)、检测线(B)、金标抗体的浓度(C)，测试其对样品检测线的影响。
(4)测试条的组装在底板上依次将包被有蛋白的硝酸纤维素膜、金标垫与玻璃纤维素膜制成的样品垫、吸水纸制成的吸收垫按图3组装，在底板上依次设置样品垫、金标垫、硝酸纤维素膜以及吸水纸，其中，硝酸纤维素膜上设置有检测区和控制区。
组装好的纸板按纵向剪切，裁成宽度为3mm的条状，即为抗体检测测试条成品。
不同NC膜型号以及控制线(A)、检测线(B)、金标抗体浓度(C)对样品检测限的影响。以表2所示的NC膜，抗小鼠抗体IgG，小鼠抗人Chemerin抗体，小鼠抗人C-23抗体免疫金的用量制备不同的测试条，并配制不同浓度的样品，以检测样品中趋化素的检测限。
表2 NC膜型号、控制线、检测线、免疫金、可检测样品最低浓度检测限D各项配比
Figure PCTCN2017116588-appb-000003
Figure PCTCN2017116588-appb-000004
通过实验结果，对小鼠抗人Chemerin抗体的量与样品最低显色检测限D用量之间的关系进行统计可知：
当检测膜选自Startorius CN95时，最低显色检测线在100-300ng/ml范围内，小鼠抗人Chemerin抗体的量B为(X±15％)μg/cm，最低显色检测限D为Y ng/ml，检测区包被的第二抗体的用量与最低显色检测限关系为Y＝-322.23X+360.79。
当检测膜选自Startorius CN140时，最低显色检测线在100-300ng/ml范围内，小鼠抗人Chemerin抗体的量B为(X±15％)μg/cm，最低显色检测限D为Y ng/ml，检测区包被的第二抗体的用量与最低显色检测限关系为Y＝-317.51X+1628.6。
当检测膜选自Pall Vivid 170时，最低显色检测线在100-300ng/ml范围内，小鼠抗人Chemerin抗体的量B为(X±15％)μg/cm，最低显色检测限D为Y ng/ml，检测区包被的第二抗体的用量与最低显色检测限关系为Y＝-309.64X+665.17。
当检测膜选自Whatman AE99时，最低显色检测线在100-300ng/ml范围内，小鼠抗人Chemerin抗体的量B为(X±15％)μg/cm，最低显色检测限D为Y ng/ml，检测区包被的第二抗体的用量与最低显色检测限关系为Y＝-316.91X+668.99。
实施例4测试条的使用方法
(1)持测试条将箭头标志的一段垂直浸入装有待测液的容器中直至箭头下端标记横线处，浸入时间为3～5秒，或者将样品滴加到样品垫上；
(2)当红色液体移行至测试区时，取出平放于干净的非吸附材料的平面。
(3)5-15分钟内判断结果，15分钟后判定无效。
检测结果说明
(1)预警值：控制线(A)和检测线(B)均出现红色条带，表示样本中chemerin浓度在该试纸条检测限以上，为高风险值。如附图5所示，若检测线(B)颜色较浅，则表明样品中chemerin浓度高，提示为高风险预警；若检测线(B)颜色较深，则表明样品中chemerin浓度较高，提示为超高风险预警。
(2)安全值：控制线(A)出现红色条带，检测线(B)颜色无色，表示样本中chemerin浓度在该试纸条检测限以下，为低风险值。
(3)无效：控制线(A)没有红色条带出现，表示测试无效。在此情况下，应重新测试。
本发明还可以采用多条不同最低检测线的试制条进行组合使用，例如，组合使用两条最低显色检测线不同的试制条，当最低显色检测线为240ng/ml的测试条的检测线(B)出现红色条带，而最低显色检测线为300ng/ml的测试条的检测线(B)未出现红色条带时，证明待测样品的中趋化素的浓度位于240ng/ml-300ng/ml之间，从而可以进一步达到半定量的目的。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种检测趋化素的测试条，其包括检测膜，检测膜的上游设置有含有以标记物标记的第一抗体的结合区，结合区上游设置有加样区，其中，检测膜上设置有检测区和控制区，控制区位于检测区下游；其中所述检测区上包被有第二抗体，所述控制区包被有抗第一抗体的二抗；所述第一抗体和第二抗体均能与趋化素特异性结合。
  2. 根据权利要求1所述的检测趋化素的测试条，结合区中包含的第一抗体选自趋化素功能性片段的抗体；优选为C-23抗体；更优选为C-23单克隆抗体，其中，C-23的序列如SEQ ID No.1所示
    CLRVQRAGEDPHSFYFPGQFAFS SEQ ID No.1。
  3. 根据权利要求1-2任一项所述的检测趋化素的测试条，所述第二抗体选自趋化素抗体。
  4. 根据权利要求1-3任一项所述的检测趋化素的测试条，所述检测膜选自硝酸纤维素膜，优选为粒径为5-20μm，爬速为80-250s/4cm；更优选为6-15μm，爬速为80-225s/4cm；最优选为①Sartorius CN95，粒径为15μm，爬速约为90-135s/4cm②Sartorius CN140，粒径为8μm，爬速约为110-165s/4cm③Pall Vivid 170，粒径为6μm，爬速为150-225s/4cm④whatman AE99，粒径为8μm，爬速约为120-160s/4cm。
  5. 根据权利要求1-4任一项所述的检测趋化素的测试条，所述检测区包被的第二抗体的用量为0.15-5.67μg/cm；
    优选地，所述检测膜选自Startorius CN95，所述检测区包被的第二抗体的用量为0.15-1.67μg/cm，待测样品最低显色检测限分别为500-60ng/ml；或者
    所述检测膜选自Startorius CN140，所述检测区包被的第二抗体的用量为4.15-5.67μg/cm，待测样品最低显色检测限分别为500-60ng/ml；或者
    所述检测膜选自Pall Vivid 170，所述检测区包被的第二抗体的用量为1.15-2.67μg/cm，待测样品最低显色检测限分别为500-60ng/ml；或者
    所述检测膜选自Whatman AE99，所述检测区包被的第二抗体的用量为 1.15-2.67μg/cm，待测样品最低显色检测限分别为500-60ng/ml；
    更优选地，所述检测膜选自Startorius CN95，所述检测区包被的第二抗体的用量为(X±15％)μg/cm，最低显色检测限为Y ng/ml，检测区包被的第二抗体的用量与最低显色检测限关系为Y＝-322.23X+360.79，最低显色检测线Y选自60-500的任意数值；或者
    所述检测膜选自Startorius CN140，所述检测区包被的第二抗体的用量为(X±15％)μg/cm，最低显色检测限为Y ng/ml，检测区包被的第二抗体的用量与最低显色检测限关系为Y＝-317.51X+1628.6，最低显色检测线Y选自60-500的任意数值；或者
    所述检测膜选自Pall Vivid 170，所述检测区包被的第二抗体的用量为(X±15％)μg/cm，最低显色检测限为Y ng/ml，检测区包被的第二抗体的用量与最低显色检测限关系为Y＝-309.64X+665.17；最低显色检测线Y选自60-500的任意数值；或者
    所述检测膜选自Whatman AE99，所述检测区包被的第二抗体的用量为(X±15％)μg/cm，最低显色检测限为Y ng/ml，检测区包被的第二抗体的用量与最低显色检测限用量关系为Y＝-316.91X+668.99，最低显色检测线Y选自60-500的任意数值；
    最优选地，所述检测区的第二抗体的用量、检测膜型号与最低显色检测限如表a所示：
    表2a
    Figure PCTCN2017116588-appb-100001
    Figure PCTCN2017116588-appb-100002
  6. 权利要求1-5所述的检测趋化素的测试条的制备方法，其包括如下步骤：
    (1)制备加样区，以样品垫处理液处理加样区；
    (2)制备结合区：以标记物标记的第一抗体，并将其涂布在结合区；
    (3)制备检测膜：稀释第二抗体及第一抗体的二抗，分别包被在检测膜上，形成检测区和控制区，干燥后以封闭液进行封闭；
    (4)组装测试条：依次将加样区、结合区、检测区和吸水垫组装至底板上。
  7. 权利要求1-5所述的检测趋化素的测试条在制备检测待测样品中趋化素的试剂盒中的用途。
  8. 权利要求1-5所述的检测趋化素的测试条在制备评估或诊断肥胖、脂肪 肝、非酒精性脂肪肝病、肝纤维化、子痫、妊娠糖尿病、糖尿病及多囊卵巢综合征的试剂盒中趋化素的试剂盒中的用途。
  9. 一种检测趋化素的方法，采用1-5任一项所述的测试条，将待测样品加到加样区，5-15分钟内根据测试条的显色判断结果；
    如果控制线无色，则测试条失效，需重新检测；如果控制线与检测线均出现红色条带，则结果为样品中趋化素浓度超过该试纸条最低显色检测限的阈值；若控制线出现红色条带，检测线无色，则结果为样品中趋化素浓度低于该试纸条最低显色检测限的阈值。
  10. 一种趋化素检测组合物，其中包含2条以上权利要求1-5任一项所述的测试条，所述测试条的最低显色检测浓度不同。
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