CN106370871A - Test paper for testing chorionic gonadotropin in human saliva and preparation method thereof - Google Patents

Test paper for testing chorionic gonadotropin in human saliva and preparation method thereof Download PDF

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CN106370871A
CN106370871A CN201610850792.0A CN201610850792A CN106370871A CN 106370871 A CN106370871 A CN 106370871A CN 201610850792 A CN201610850792 A CN 201610850792A CN 106370871 A CN106370871 A CN 106370871A
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pad
sample
sample pad
nitrocellulose membrane
hcg
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CN201610850792.0A
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Chinese (zh)
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马北峰
马北阳
徐晓峰
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武汉百美生物科技有限公司
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Priority to CN201610850792.0A priority Critical patent/CN106370871A/en
Publication of CN106370871A publication Critical patent/CN106370871A/en

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/76Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/36Gynecology or obstetrics

Abstract

Provided is a test paper for testing chorionic gonadotropin in human saliva. The test paper comprises a primary sample pad, nitrocellulose filter membrane with testing T line and quality control C line, a sample suction pad and a bottom plate. The primary sample pad, the nitrocellulose filter membrane and the sample suction pad are respectively connected with the bottom plate through lap joints to form a test paper; among which the upper side of the primary sample pad is pasted with a secondary sample pad for filtering the impurities in the sample. The test paper has the advantages of 1, the design of the secondary sample pad in both single and double channel test papers can effectively filter the impurities in the saliva, especially the mucoprotein, miscellaneous bacteria and the like; 2, the sample in the double channel test paper passes through a first level channel to touch the test line without creating any composite substance through the combination with labeled antibody in the binding pad, the labeled antibody passes through a second level channel and touches the test line. This test paper overcomes the concentration/coagulation problem between the labeled sample and sample analyte.

Description

一种检测人唾液中绒毛膜促性腺激素的试纸及其制备方法 One kind of paper and preparation method of human chorionic gonadotropin saliva Detection

技术领域 FIELD

[0001] 本发明涉及体外免疫诊断试剂类技术领域,具体地说是一种检测人唾液中绒毛膜促性腺激素的试纸及其制备方法。 [0001] The present invention relates to in vitro immunodiagnostic reagents technical field, particularly in human saliva test strip for detecting hCG and its preparation method.

背景技术 Background technique

[0002] 育龄女性妊娠时体内胎盘滋养层细胞会分泌一种激素,称为人绒毛膜促性腺激素(Human Chorionic Gonadotrophin,简称HCG)。 [0002] Women of childbearing age during pregnancy trophoblast cells in vivo secrete a hormone known as human chorionic gonadotropin (Human Chorionic Gonadotrophin, referred to as HCG). 它是一种由〇和0二亚基组成的糖蛋白激素, 其中a亚基与垂体分泌的卵泡刺激素(FSH)、黄体生成素(LH)和促甲状腺激素(TSH)等基本相似,故相互间能发生交叉反应,而ME基的结构各不相同。 It is a binary 0 and a square glycoprotein hormone subunits, where a subunit of pituitary follicle-stimulating hormone (FSH), luteinizing hormone (LH) and thyroid stimulating hormone (TSH) and the like are substantially similar, it is can cross-react with each other, and the structure of ME group varies. HCG主要功能就是刺激黄体,有利于雌激素和黄体酮持续分泌,以促进子宫蜕膜的形成,使胎盘生长成熟。 HCG main function is to stimulate the corpus luteum, facilitate secretion of estrogen and progesterone continue to facilitate the formation of the decidua, placenta mature grown. 成熟女性因受精卵子移动到子宫腔内着床后,形成胚胎,在发育成长为胎儿过程中,胎盘合体滋养层细胞产生大量HCG,可通过孕妇血液循环而排泄到尿中。 Mature women because of the fertilized ovum moves into the uterine cavity implantation, the embryo is formed, during fetal placental syncytiotrophoblast cells produce large amounts of HCG in the growth and development, it can be excreted into the urine through the blood circulation of pregnant women. 胎盘、血液和尿中均存在HCG及其代谢产物。 Placenta, blood and urine HCG and its metabolites are present. 在受孕后7-10天左右,就可以在孕妇的晨尿中检测到HCG。 About 7-10 days after conception, HCG can be detected in the morning urine of pregnant women. 正常妊娠妇女血清中,受精卵植入子宫后的4-7天后开始增加,并以指数形式增长,平均每1.5-2天浓度就翻倍。 Women in serum of normal pregnancy, the fertilized egg implanted in the uterus after 4-7 days began to increase, and increased exponentially, on average every 1.5 days concentration doubled. HCG是早期妊娠中的主要信号及特异性标志,已成为临床上诊断早期妊娠的常用指标,被广泛应用于早期妊娠的临床诊断及家庭自我检测,如目前使用的检测人HCG的检测工具为血或尿HCG 胶体金免疫层析检测试纸,它能在几分钟~十几分钟内完成,具有安全、费用低廉、易保存、 稳定性好、快速、应用方便以及阳性率较高等优点而被广泛应用。 HCG is the main signal early pregnancy and specific markers, has become a commonly used index clinical diagnosis of early pregnancy, early pregnancy is widely used in clinical diagnosis and home self-testing, as currently used to detect human blood HCG testing tools or urine HCG GICA test strip, which can be completed in minutes - ten minutes, with a safe, low cost, easy to preserve, good stability, fast, easy to use and high positive rate, etc. have been widely used . 目前市面上传统的试纸条主要通过测试尿液来判断是否怀孕,但尿试纸仍存在一些局限性如需要采集尿液且不够洁净,如操作不当容易造成污染;大量不同的文献报道检测的敏感性(阳性率)仅为62-78%, 假阳性率高达26-40%。 Currently on the market mainly through traditional test strip urine test to determine whether the pregnancy, but the urine test paper there are still some limitations such as the need to collect urine and clean enough, such as improper operation likely to cause pollution; a lot of different literature sensitive detection of (positive rate) of only 62-78% false positive rate as high as 26-40%. 血试纸敏感性、特异性高,假阳性也少,但是如果作为早期妊娠的即时诊断或家庭诊断方法,也存在刺痛、采血不方便、需要到专业医院就诊等问题,育龄女性在选择自我或即时诊断早孕时很少使用。 Blood test strips sensitivity, high specificity, false positives less, but if the family as an instant diagnosis or diagnosis of early pregnancy, there is tingling, blood collection is not convenient, you need to specialty hospital issues such as women of childbearing age in the choice of self or instant diagnosis is rarely used during pregnancy. 如果能找到血试纸一样高的敏感性、特异性,又无尿试纸的局限性,将大大方便即时诊断育龄女性的早期妊娠。 If you can find as high a blood test strips sensitivity, specificity, nor the limitations of the urine test paper, will greatly facilitate the immediate diagnosis of early pregnancy, women of childbearing age.

[0003] 人体的唾液检测符合这一要求。 [0003] saliva to detect human body meets this requirement. 唾液是人体口腔内的唾液腺分泌的液体和口腔上的许多小腺体所分泌的粘液在口腔内混合而成,正常人每天分泌唾液约1~1.5升,为白色无味液体,除水分外含有大量有机物、维生素、氨基酸、粘蛋白、球蛋白、淀粉酶、溶菌酶、血型物质、口腔粘膜上皮细胞等物质。 Saliva salivary secretion in the human oral cavity and many small liquid oral glands secrete mucus mixed in the oral cavity, the normal secretion of saliva per day about 1 to 1.5 liters, a white odorless liquid, in addition to containing a large amount of water organics, vitamins, amino acids, mucin, globulin, amylase, lysozyme, blood group substances, oral mucosa epithelial cells and other substances. 选择采集唾液的部位为牙龈组织,所收集的多为口腔粘膜漏出液,且多采用如擦拭法收集,病人或体检者自己都可以收集,可以在任何时间、任何地点收集。 Selecting a portion of saliva collected gum tissue, mostly collected oral mucosal transudate, such as collecting and using multi-wiping method, the patient or medical themselves can be collected, it can be collected at any time, any place. 唾液的作用可不只局限在润滑口腔、保护牙齿和帮助消化,它和尿液、血液一样, 可用于疾病诊断。 The role of saliva can not just confined to lubricate the mouth to protect teeth and help digestion, and it is urine, blood, like, can be used for disease diagnosis. 众所周知,唾液的成分与血液成分极其相似,理论上通过血液能查出的疾病,唾液也能检测到。 As we all know, saliva composition and blood components is very similar, in theory, be able to detect the disease through blood, saliva can be detected. 用唾液诊断疾病并不罕见,美国食品药品监督管理局早在2003年就批准通过唾液快速检测艾滋病病毒(HIV);国内目前也已经有同类产品上市,如由北京玛诺生物制药有限公司在国内生产的"爱卫"牌唾液测艾滋产品,这种测试盒只需20分钟,就可以帮助医生作出诊断,准确率超过99%。 With saliva diagnosis of the disease is not uncommon, the US Food and Drug Administration as early as 2003 to approve the rapid detection of the AIDS virus (HIV) through saliva; currently there are already domestic similar products on the market, such as the Beijing Biological Pharmaceutical Co., Ltd. in the domestic Shimano production of "Ai Wei" brand products saliva HIV test, this test box just 20 minutes, it can help doctors make a diagnosis, the accuracy rate of over 99%. 常规的HIV血检测法通常需要2个星期才能出结果,这种方法可以让更多的高危人群进行测试,包括那些对取指血害怕的人,将消除人们的紧张和恐惧心理,对人体也是安全的。 Routine HIV blood assay usually takes two weeks to the results, this method can allow more testing high-risk groups, including those of human blood fetch afraid, will dispel the tension and fear, but also on the human body safe.

[0004] 业内发现,正常月经周期中,甾体性激素在唾液中的水平与外周血中水平显著相关,故唾液标本可代替血标本测定留体激素,了解黄体期子宫的激素内环境。 [0004] Others found that the normal menstrual cycle, the level of the steroid hormone levels in peripheral blood were significantly associated saliva, sputum samples can be determined so that steroidal hormones, uterine Learn luteal phase hormonal environment instead of blood samples. 但是目前未发现有应用纳米材料免疫层析试纸法检测唾液中的HCG、HCG-|3,特别是应用纤维素纳米颗粒、 亲和素、链霉生物素的免疫层析试纸法检测唾液中的HCG、HCGj。 However, there is not currently found nanomaterials immunochromatographic strip assay saliva HCG, HCG- | 3, applied in particular cellulose nanoparticles, avidin, streptavidin immunochromatography strip assay saliva HCG, HCGj. 由于唾液中HCG、HCG-的则得值低于血测得值,不同的研究学者报道约达10-20倍差异。 Because saliva HCG, HCG- is obtained value is lower than the measured value of the blood, different researchers reported that about 10 to 20 fold difference. 如果检测唾液中HCG、HCG-|3,不选用费时、费力和成本高的放射免疫法和酶免疫法,而选择方便、快捷,能作为即时诊断的纳米材料免疫层析试纸检测法,必将大大方便早期妊娠的自我检测,这就要求纳米材料免疫层析试纸法检测唾液中HCG、HCG-邱勺灵敏度要高、特异性要强、阳性率高、假阳性率要低, 其微量的检测功能还需符合血或尿试纸的质量标准要求,才能很好地应用于早期妊娠的即时诊断。 If it is detected in saliva HCG, HCG- | 3, do not use time-consuming, laborious and costly radioimmunoassay and enzyme immunoassay, and choose convenient, fast and can be used as nano-materials immunochromatographic dipstick assay instant diagnosis, will early pregnancy greatly facilitate the self-test, which requires nanomaterials immunochromatographic strip assay saliva HCG, HCG- Qiu spoon higher sensitivity, specificity stronger positive rate, false positive rate is lower, which trace a detection function the need to meet the quality standard blood or urine test paper requirements in order to be well applied to the instant diagnosis of early pregnancy. 目前尿HCG试纸多是运用胶体金免疫双抗体夹心的方法,这种HCG胶体金免疫层析检测试纸是无法达到这一水平的,特别是在唾液非检测物干扰、灵敏度等方面。 Currently is the use of multiple dipstick urine HCG immunogold double antibody sandwich method, such HCG gold immunochromatography test strips can not achieve this level, especially in the saliva of non-interference was detected, the sensitivity and the like.

发明内容 SUMMARY

[0005] 本发明的目的是提供一种检测人唾液中绒毛膜促性腺激素的试纸及其制备方法, 运用双抗体夹心免疫层析技术,实现对被检样本唾液中绒毛膜促性腺激素的检测,它方便、 快捷、准确性高,特别适合于育龄女性对早期妊娠的即时诊断。 [0005] The object of the present invention is to provide a method for detecting human saliva test paper and preparation of hCG, using double antibody sandwich immunochromatography technique, detection of the test sample to achieve saliva hCG it convenient, fast, high-accuracy, especially for women of childbearing age immediate diagnosis of early pregnancy.

[0006] 本发明一方面提供一种检测人唾液中绒毛膜促性腺激素的试纸,包括:第一样本垫、包被有检测T线与质控C线的硝酸纤维素膜、吸样垫、底板,所述第一样本垫、所述硝酸纤维素膜、所述吸样垫依次搭接于所述底板,构成一检测试纸; [0006] In one aspect the present invention provides a method of detecting human saliva test paper hCG, comprising: a first sample pad, coated with a nitrocellulose membrane detected T-line quality control and C-line, aspirating pad , the bottom plate, the first sample pad, a nitrocellulose membrane, said pad aspirating sequentially overlapped to the base plate constitutes a test strip;

[0007] 其中,所述第一样本垫上方粘贴一第二样本垫,用于过滤样品中杂质。 [0007] wherein the first sample a second sample pad on the adhesive pad, for filtering impurities in the sample.

[0008] 还包括两端均粘贴于所述硝酸纤维素膜上的结合垫,且所述第一样本垫一端搭接于所述结合垫上方,使得所述第一样本垫、所述第二样本垫、所述结合垫、所述硝酸纤维素膜、所述吸样垫、所述底板共同构成一单通道试纸。 [0008] further comprises at both ends attached to the cellulose nitrate film, bond pads, and overlapping an end of the first sample pad to the bonding pad side, such that the first sample pad, the a second sample pad, a conjugate pad, a cellulose nitrate film, the aspirating pad, said bottom plate together constitute a single channel strip.

[0009] 或者试纸包括上下依次层叠的结合垫、纤维隔离薄膜,所述纤维隔离薄膜两端分别搭接于所述第一样本垫、所述硝酸纤维素膜上方,且所述结合垫一端与所述纤维隔离薄膜的一侧平齐,另一端从所述纤维隔离薄膜的另一侧延伸、搭接于所述硝酸纤维素膜上,使得所述第一样本垫、所述第二样本垫、所述结合垫、所述纤维隔离薄膜、所述硝酸纤维素膜、 所述吸样垫、所述底板构成双通道试纸。 [0009] The strip comprises upper and lower stacking or bond pads, fibrous insulation film, both ends of the fibrous insulation film are overlapped to the first sample pad, nitrocellulose membrane is upward, and one end of the bonding pads side of the fibrous insulation film is flush with the other end extending from the other side of the fibrous insulation film, cellulose nitrate film overlapping, such that the first sample pad, the second sample pad, a conjugate pad, the fibrous insulation film, cellulose nitrate film, the aspirating pad, a 2-channel strip the floor.

[0010] 本发明另一方面提供所述第一方面试纸的制备方法,包括如下步骤: [0010] In another aspect of the present invention provides a first aspect of the method of preparing paper comprising the steps of:

[0011] SI:样本垫的制备 [0011] SI: Preparation of the sample pad

[0012] 第一样本垫的制备:将剪裁后的玻璃纤维膜、或无纺布、或过滤试纸置于第一样本垫处理液中室温浸泡、烘干、备用; [0012] Preparation of the first sample pad: glass fiber film after trimming, or nonwoven fabric, or filter paper placed in a first sample pad soaked in the treatment liquid at room temperature, drying, standby;

[0013] 第二样本垫的制备:选取孔径为500nm-1000um过滤试纸、或玻璃纤维膜、或无纺布、或吸水滤纸,在浓度为〇. I -I 〇〇mg/ml的羟基磷灰石液体中室温浸泡、烘干、备用; [0013] Preparation of the second sample pad: Select a pore size of 500nm-1000um filter paper, glass fiber or film, or a nonwoven fabric, or absorbent filter paper square in a concentration of hydroxyapatite I -I 〇〇mg / ml. stone immersion liquid at room temperature, drying, standby;

[0014] S2:结合垫的制备:配制结合垫处理液,标记抗体,将结合垫原料置于结合垫处理液中室温浸泡、烘干,再用划膜喷金机将纤维素纳米标记抗体或金标抗体或二者的混合物喷于经浸泡、烘干后的结合垫原料上、再烘干备用; [0014] S2: Preparation of pad binding: formulating pad binding process liquid, labeled antibody, the bound material placed on the pad conjugate pad soaked in the treatment liquid at room temperature, dried, and then draw the film spraying machine labeled antibody or cellulose nano or gold-labeled antibody mixture was sprayed on both soaked, the pad binding material after drying, and then drying the standby;

[0015] 且制得的所述结合垫上有150-550nm纤维素纳米颗粒,10-80nm胶体金或者二者的混合物,且标记有HCG抗体a、P特异性抗体或者抗完整HCG抗体;或有乳胶颗粒或碳颗粒或其它发光染料或酶,且结合有HCG抗体a、辦寺异性抗体或者抗完整HCG抗体,或引用或结合亲和素、生物素。 [0015] and the conjugate pad made of cellulose nanoparticles have 150-550nm, 10-80nm colloidal gold or a mixture of the two, and HCG antibody labeled with a, P specific antibodies or an anti-HCG antibody complete; or latex particles or carbon particles or other luminescent dye or an enzyme, and an antibody bound HCG a, or anti-heterophilic antibodies do temple complete HCG antibody, or reference, or avidin binding, biotin.

[0016] S3:硝酸纤维素膜包被 [0016] S3: coated nitrocellulose membrane

[0017] 配制包被缓冲液,用包被缓冲液分别稀释羊抗鼠多克隆抗体、抗a-HCG或FHCG的抗体,或者抗完整HCG抗体,获得质控C线和检测T线; [0017] The formulated coating buffer, with coating buffer were diluted goat anti-mouse polyclonal antibody, or a-HCG antibodies FHCG or complete anti-HCG antibodies obtained quality control line and testing line T C;

[0018] 且所述硝酸纤维素膜的检测T线上可设置有亲和素、生物素。 [0018] and detects the line nitrocellulose membrane is T may be provided with avidin, biotin.

[0019] S4:试纸的制备 S4 [0019]: Preparation of test strips

[0020] 单通道试纸的制备:在常温的干燥室内,取底板,先将包被的硝酸纤维素膜贴合于底板中部,将结合垫粘贴于硝酸纤维素膜上方,再将第一样本垫一端搭接于结合垫上方,另一端与底板一侧平齐;然后将吸样垫一端搭接于硝酸纤维素膜上方,另一端与底板另一侧平齐后,将第二样本垫粘贴于第一样本垫上方,最后采用裁剪机剪切底板后形成试纸; [0020] Preparation of single-channel test strips: room temperature in the drying chamber, to take the floor, first coated nitrocellulose membrane in its central portion is bonded to the above bond pads attached to a nitrocellulose membrane, and then the first sample One end of the lap pad to the conjugate pad side, and the other end is flush with the bottom plate side; then aspirating pad overlapped one end of the above nitrocellulose membrane, and the other end is flush with the other side of the base plate, the second paste sample pad sample pad to the first party, after the paper is formed using the last shear cutter plate;

[0021] 双通道试纸的制备:在常温的干燥室内,取底板,先将包被的硝酸纤维素膜贴合于底板中部,将第一样本垫与吸样垫搭接于硝酸纤维素膜两端上方,且第一样本垫、吸样垫远离硝酸纤维素膜的一端均与底板平齐;第一样本垫上方粘贴第二样本垫,使得待测样本依次通过第二样本垫、第一样本垫、硝酸纤维素膜、吸样垫形成第一水平流动通道; [0021] Preparation of a dual-channel strip: the drying chamber at room temperature, to take the floor, first coated nitrocellulose membrane in its central portion is bonded to the first aspirating the sample pad and the nitrocellulose membrane pad overlapped upper ends, and the first sample pad, the sample pad away from the suction end of the nitrocellulose membrane are flush with the bottom plate; a first adhesive pad on a second sample of the sample pad, so that the test sample passes through the second sample pad, The first sample pad, a nitrocellulose membrane, the sample pad is formed of a first level of suction flow passage;

[0022] 将纤维隔离薄膜两端分别搭接在第一样本垫、硝酸纤维素膜上,结合垫粘贴于纤维隔离薄膜上方,且结合垫一端与纤维隔离薄膜的一侧平齐,另一端从纤维隔离薄膜的另一侧延伸、搭接于硝酸纤维素膜上,使得检测过程中所加的缓冲液体依次通过结合垫、纤维隔离薄膜、硝酸纤维素膜、吸样垫形成第二水平流动通道,最后采用裁剪机剪切底板后形成双通道试纸。 [0022] The ends of the fiber are overlapped in the first separating membrane sample pad, a nitrocellulose membrane, above the bond pads attached to a fibrous insulation film, and one end side bond pads flush with the fibrous insulation film, and the other end It extends from the other side of the fibrous insulation film, overlapping on nitrocellulose filters, so that the detection process of adding the liquid passes through binding buffer pad, fibrous insulation film, cellulose nitrate film, forming a second pad aspirating horizontal flow channel, after the last shear cutter plate is formed using a dual-channel strip.

[0023] -种检测人唾液中绒毛膜促性腺激素的试纸及其制备方法,其优点是: [0023] - saliva test strip detection of human chorionic gonadotropin species and its preparation method, its advantages are:

[0024] ①、单、双通道试纸中第二样本垫的设计,有效过滤唾液中的杂质,特别是粘蛋白、 杂菌等; [0024] ①, mono, two-channel second sample pad test strip design, saliva effectively filter impurities, especially mucin, bacteria and the like;

[0025] ②、双通道试纸中样本通过第一水平通道达到检测线,且不与结合垫中标记抗体结合而产生复合物,标记抗体通过第二水平通道迀移达到检测线,克服了标记物与样本中分析物之间的聚集/凝聚问题; [0025] ②, two-channel test strip by the sample reaches the first horizontal passage detection line, and does not bind the labeled antibody binds to produce the composite pad, a second labeled antibody by detecting the horizontal channel Gan shift reaches line, overcomes the marker and aggregation between the analyte in the sample / agglomeration problems;

[0026] ③、通过示踪标志物纤维素纳米标记抗体或金标抗体或二者的混合物,或者乳胶颗粒或碳颗粒或其它发光染料或酶结合的抗a-HCG或P-HCG的抗体或者抗完整HCG抗体的选择,以及结合垫、硝酸纤维素膜中亲和素、生物素的应用,大大提高了示踪标志物的良好标记,同时也增强或放大了检测信号,克服了单纯胶体金标记抗体的免疫层析检测试纸,其敏感性(阳性率)较低以及假阳性率高的局限性,从而体现出本发明的高特异、假阳性率低和高敏感性,实现对被检样本人唾液中绒毛膜促性腺激素(HCG)的检测,与尿HCG的检测试纸比较,它更清洁、方便、快捷,特别适合于育龄女性对早期妊娠的即时诊断。 [0026] ③, by tracer antibody labeled marker substance or a mixture of cellulose nano-gold labeled antibody, or both, or latex particles or carbon particles or other light emitting dyes, or enzyme-conjugated anti-a-HCG or P-HCG antibody, or complete anti-HCG antibody selection, and conjugate pad, a nitrocellulose membrane, avidin, biotin, greatly improving a good marker labeled tracer, but also enhances or amplifies the detection signal, overcome the simple colloidal gold antibody labeled immunochromatographic test strip, the sensitivity (positive rate) and a low false positive rate of limitations, which reflects the high specificity of the present invention, high sensitivity and lower false positive rate, subject the sample to achieve the human saliva chorionic gonadotropin (HCG) testing, compared with the urine HCG test paper, it is more clean, convenient, fast, especially for women of childbearing age immediate diagnosis of early pregnancy.

附图说明 BRIEF DESCRIPTION

[0027]图1为本发明单通道结构示意图; [0027] FIG. 1 is a schematic single-channel structure of the present invention;

[0028]图2为本发明双通道结构示意图; [0028] FIG. 2 is a schematic view of a dual-channel structure of the invention;

[0029]图3为本发明试纸的外壳结构示意图; [0029] FIG. 3 is a schematic of the test strip housing structure of the present invention;

[0030] 其中: [0030] wherein:

[0031] I、第一样本垫, [0031] I, the first sample pad,

[0032] 2、硝酸纤维素膜,21、检测T线,22、质控C线,23、硝酸纤维素膜视窗, [0032] 2, a nitrocellulose membrane, 21, line T is detected, 22, the C-line quality control, 23, nitrocellulose membrane window,

[0033] 3、吸样垫,31、吸样垫视窗, [0033] 3, the sample suction pad 31, absorbent pad-like window,

[0034] 4、底板, [0034] 4, the bottom plate,

[0035] 5、第二样本垫,51、加样孔, [0035] 5, a second sample pad 51, sample hole,

[0036] 6、结合垫,52、加液(试剂)孔 [0036] 6, bonding pads 52, and fluid (agent) hole

[0037] 7、纤维隔离薄膜, [0037] 7, fibrous insulation film,

[0038] 8、外壳。 [0038] 8, the housing.

具体实施方式 Detailed ways

[0039] 根据图1、图2所示:一种检测人唾液中绒毛膜促性腺激素的试纸,包括:第一样本垫1、包被有检测T线21与质控C线22的硝酸纤维素膜2、吸样垫3、底板4,所述第一样本垫1、 所述硝酸纤维素膜2、所述吸样垫3依次搭接于所述底板4,构成一检测试纸; [0039] According to FIG. 1, FIG. 2: A method of detecting human chorionic gonadotropin saliva test strip, comprising: a first sample pad 1, are coated with nitric acid 22 21 T-line detection and quality control line C cellulose membrane 2, aspirating pad 3, the base plate 4, the first sample pad 1, nitrocellulose membrane is 2, the aspirating pad 3 sequentially overlapped to the base plate 4 to form a test strip;

[0040] 其中,所述第一样本垫1上方粘贴一第二样本垫5,用于过滤样品中杂质。 [0040] wherein the first sample a second pad attached above a sample pad 5, for filtering impurities in the sample.

[0041] 优选地,试纸还包括两端均粘贴于所述硝酸纤维素膜2上的结合垫6,且所述第一样本垫1 一端搭接于所述结合垫6上方,使得所述第一样本垫1、所述第二样本垫5、所述结合垫6、所述硝酸纤维素膜2、所述吸样垫3、所述底板4共同构成一单通道试纸。 [0041] Preferably, further comprising a strip attached at both ends to a nitrocellulose membrane is bound on the pad 2 6, 1 and the first end overlaps sample pad to the upper pad binding 6, such that the the first sample pad 1, the second sample pad 5, the bonding pad 6, the cellulose nitrate film 2, the aspirating pad 3, the base plate 4 together constitute a single channel strip.

[0042] 优选地,试纸还包括上下依次层叠的结合垫6、纤维隔离薄膜7,所述纤维隔离薄膜7两端分别搭接于所述第一样本垫1、所述硝酸纤维素膜2上方,且所述结合垫6-端与所述纤维隔离薄膜7的一侧平齐,另一端从所述纤维隔离薄膜7的另一侧延伸、搭接于所述硝酸纤维素膜2上,使得所述第一样本垫1、所述第二样本垫5、所述结合垫6、所述纤维隔离薄膜7、所述硝酸纤维素膜2、所述吸样垫3、所述底板4构成双通道试纸。 [0042] Preferably, the test strip further comprises upper and lower bonding pads 6 are sequentially stacked, fibrous insulation film 7, the fibrous insulation films 7 are respectively overlapped on both ends of the first sample pad 1, the cellulose nitrate film 2 above, and the end of the bond pads 6- fibrous insulation film 7 side is flush with the other end extending from the other side of the fibrous insulation film 7, the lapped on a nitrocellulose membrane 2, 1 such that the first sample pad, the second sample pad 5, the bonding pad 6, the fibrous insulation film 7, the cellulose nitrate film 2, the aspirating pad 3, the base plate 4 a 2-channel strip.

[0043] 优选地,所述第一样本垫1由玻璃纤维膜或无纺布构成;所述吸样垫3由吸水滤纸构成; [0043] Preferably, the first sample 1 is made of a glass fiber mat film or a nonwoven fabric; the absorbent sample pad 3 is made of water-absorbing filter paper;

[0044] 进一步地,所述第二样本垫5为500nm-1000um孔径的过滤试纸、或玻璃纤维膜、或无纺布或吸水滤纸,且所述第二样本垫5含有0.1-100mg/ml浓度的羟基磷灰石; [0044] Further, the second sample pad 5 is 500nm-1000um pore size filter paper, glass fiber or film, or a nonwoven fabric or a water-absorbing filter paper pad 5 and the second sample contained 0.1-100mg / ml concentration hydroxyapatite;

[0045] 所述结合垫6中含150-550nm纤维素纳米颗粒、或10-80nm胶体金,或二者的混合物,且所述结合垫6中结合有抗a-HCG或P-HCG的抗体或者抗完整HCG抗体; [0045] In the bonding pads 6, or a mixture of both, or colloidal gold 10-80nm 150-550nm cellulose-containing nanoparticles, and the a-HCG antibody, or anti-P-HCG 6 in conjunction with bond pads or complete anti-HCG antibody;

[0046] 进一步地,所述结合垫6中有乳胶颗粒或碳颗粒或其它发光软染料或酶,且结合有抗a-HCG或P-HCG的抗体或者抗完整HCG抗体,或引用或结合亲和素、生物素; [0046] Further, the bonding pad 6 latex particles or carbon particles or other soft luminescent dye or an enzyme, and the bound a-HCG or anti-P-HCG antibody, or anti-HCG antibodies complete, or reference, or binding affinity and biotin;

[0047] 优选地,所述硝酸纤维素膜2的检测T线21上设置有抗a-HCG或fHCG的抗体或者抗完整HCG抗体; [0047] Preferably, the nitrocellulose membrane is provided with a-HCG antibodies or fHCG or anti-HCG antibody 21 detects the full line T 2;

[0048] 所述硝酸纤维素膜2膜的质控C线22上设置有羊抗鼠多克隆隆抗体; [0048] provided with a goat anti-mouse antibody Duokelonglong 2 22 C-line quality control of the nitrocellulose membrane;

[0049] 所述硝酸纤维素膜2上的检测T线21上也可设置亲和素、生物素。 The [0049] T is detected on the line 2 on the nitrocellulose membrane 21 may be provided avidin, biotin.

[0050] 上述中的抗(1-1«1;或|3-1«1;的抗体,或者抗完整1«1;抗体的用量在0.01-10011^/1111。 [0050] Anti above (1-1 << 1; or | 3-1 << 1; antibody, or an anti complete «1; the amount of antibodies in the 0.01-10011 ^ / 1111.

[0051] 本发明另一方面提供所述第一方面试纸的制备方法,包括如下步骤: [0051] The present invention provides a first aspect of the aspect of the production method of the strip, comprising the steps of:

[0052] SI:样本垫的制备 [0052] SI: Preparation of the sample pad

[0053] 第一样本垫1的制备:将剪裁后的玻璃纤维膜、或无纺布、或过滤试纸置于第一样本垫1处理液中室温浸泡、烘干、备用; [0053] The first sample pad prepared in 1: glass fiber film after trimming, or nonwoven fabric, or filter paper placed in a first sample pad soaked in a treatment solution at room temperature, drying, standby;

[0054] 进一步地,第一样本垫1处理液的配制方法为:称取0.5g±0.05g BSA于烧杯中,加入70ml IOmM pH8.4硼酸盐缓冲液溶解,加入吐温-20,混匀,定容、滤膜过滤后备用。 [0054] Further, the method of the first sample pad prepared as a treatment solution: Weigh 0.5g ± 0.05g BSA in beaker, add 70ml IOmM pH8.4 borate buffer solution, was added Tween-20, mix, volume, membrane filtration after use.

[0055] 第二样本垫5的制备:选取孔径为500nm-1000um过滤试纸、或玻璃纤维膜、或无纺布、或吸水滤纸,在浓度为〇. I -I 〇〇mg/ml的羟基磷灰石液体中室温浸泡、烘干、备用; [0055] 5 second sample pad prepared: Select a pore size of 500nm-1000um filter paper, glass fiber or film, or a nonwoven fabric, or absorbent filter paper square in a concentration of I -I 〇〇mg / ml of hydroxyapatite. Greystone immersion liquid at room temperature, drying, standby;

[0056] 进一步地,所述第一样本垫1、所述第二样本垫5制备过程中的浸泡时间均为120土51^11,烘干温度均为37°。 [0056] Further, the first sample pad 1, the soaking time during the preparation of the second sample pad 5 are in the soil 51 120 ^ 11, the drying temperatures are 37 °.

[0057] S2:结合垫6的制备:配制结合垫6处理液,标记抗体,将结合垫6原料置于结合垫6 处理液中室温浸泡4h、烘干,再用划膜喷金机将纤维素纳米标记抗体或金标抗体或二者的混合物喷于经浸泡、烘干后的结合垫6原料上、再烘干备用; [0057] S2: Preparation of bond pads 6: 6 treatment liquid formulation pad binding, labeled antibody, the binding bond pads 6 disposed feed treatment liquid immersed pads 6 4h, drying at room temperature, and then spraying machine zoned film fibers the nanometer gold labeled antibody or a labeled antibody or a mixture of both soaked in spray, with the pad after drying the raw material 6, and then drying the standby;

[0058] 结合垫6处理液的制备方法:称取0.2g ± 0.05g酪蛋白、0.5g ± 0.05g海藻糖于烧杯中,加入70ml IOmM pH8.0硼酸盐缓冲液溶解,加入吐温-20,混匀、滤膜过滤后备用。 Preparation Method [0058] bonding pads 6 treatment solution: Weigh 0.2g ± 0.05g casein, 0.5g ± 0.05g trehalose in beaker, add 70ml IOmM pH8.0 borate buffer solution, was added Tween - 20, mixing, membrane filtration after use.

[0059] S3:硝酸纤维素膜2包被 [0059] S3: 2 coated nitrocellulose membrane

[0060] 配制包被缓冲液,用包被缓冲液分别稀释羊抗鼠多克隆抗体、抗a-HCG或FHCG的抗体,或者抗完整HCG抗体,获得质控C线22和检测T线21; [0060] The formulated coating buffer, with coating buffer were diluted goat anti-mouse polyclonal antibody, or a-HCG antibodies FHCG or complete anti-HCG antibodies obtained C line quality control line 22 and detector 21 T;

[0061] 进一步地,包被缓冲液的制备方法为:取海藻糖于烧杯中,加入PBS溶解后再加入甲醇,混匀、定容、滤膜过滤后备用。 Preparation Method [0061] Further, as coating buffer: Trehalose taken in a beaker, dissolved in PBS was added and then methanol was added, mixed, volume, membrane filtration after use.

[0062] S4:试纸的制备 S4 [0062]: Preparation of test strips

[0063] 单通道试纸的制备:在常温的干燥室内,取底板4,先将包被的硝酸纤维素膜2贴合于底板4中部,将结合垫6粘贴于硝酸纤维素膜2上方,再将第一样本垫1 一端搭接于结合垫6 上方,另一端与底板4一侧平齐;然后将吸样垫3-端搭接于硝酸纤维素膜2上方,另一端与底板4另一侧平齐后,将第二样本垫5粘贴于第一样本垫1上方,最后采用裁剪机剪切底板4 后形成试纸; [0063] Preparation of single-channel test strips: room temperature in the drying chamber, to take the floor 4, first coated nitrocellulose membrane 2 is bonded to the middle plate 4 and the bonding pad 6 attached to a nitrocellulose membrane 2 above, and then the first sample pad to a conjugate pad overlapped one end of the top 6, and the other end is flush with one side of the base plate 4; then aspirating end of the lap pad 3- above nitrocellulose membrane 2, the other end of the other plate 4 after one side of the flush, a second sample pad 5 attached to the top of the first sample pad 1, the shearing Finally, the bottom plate cutting strip 4 is formed;

[0064] 双通道试纸的制备:在常温的干燥室内,取底板4,先将包被的硝酸纤维素膜2贴合于底板4中部,将第一样本垫1与吸样垫3搭接于硝酸纤维素膜2两端上方,且第一样本垫1、 吸样垫3远离硝酸纤维素膜2的一端均与底板4平齐;第一样本垫1上方粘贴第二样本垫5,使得待测样本依次通过第二样本垫5、第一样本垫1、硝酸纤维素膜2、吸样垫3形成第一水平流动通道; [0064] Preparation of a dual-channel strip: the drying chamber at room temperature, taking the floor 4, first coated nitrocellulose membrane 2 bonded to the middle plate 4, the first absorbent sample pad and the loading pad 3 1 lap both ends of the above nitrocellulose membrane 2, and the first sample pad 1, pad aspirating end remote from the nitrocellulose membrane 3 2 4 are flush with the bottom plate; a first sample pad 1 attached over the second sample pad 5 , so that a second test sample passes through the sample pad 5, a first sample pad, a nitrocellulose membrane 2, the suction pad 3 is formed like a first horizontal flow channel;

[0065] 将纤维隔离薄膜7两端分别搭接在第一样本垫1、硝酸纤维素膜2上,结合垫6粘贴于纤维隔离薄膜7上方,且结合垫6-端与纤维隔离薄膜7的一侧平齐,另一端从纤维隔离薄膜7的另一侧延伸、搭接于硝酸纤维素膜2上,使得检测过程中所加的缓冲液体依次通过结合垫6、纤维隔离薄膜7、硝酸纤维素膜2、吸样垫3形成第二水平流动通道,最后采用裁剪机剪切底板4后形成双通道试纸。 [0065] The fibrous insulation film 7 overlaps both ends at a first sample pad 1, 2, 6 bond pads attached to nitrocellulose membrane fibrous insulation film 7 above, 6- and 7 end with a fibrous insulation film bonding pads flush with the side, the other end extending from the other side of the fibrous insulation film 7, lap nitrocellulose film 2, so that the detection process of liquid buffer applied through the bond pad 6 sequentially, fibrous insulation film 7, nitrate cellulose membrane 2, the pad 3 is formed aspirating a second horizontal flow channels, Finally, the bottom plate cutting machine shear strip 4 forms a double passage.

[0066] 上述试纸外还设置有一外壳8,且外壳8上设计有与第二样本垫5、结合垫6、硝酸纤维素膜2上检测T线21和质控C线22、吸样垫3依次对应设置的加样孔51、加液(试剂)孔52、硝酸纤维素膜视窗23、吸样垫视窗31。 [0066] the above paper is also provided with a housing 8, and the design of the housing 8 and the 5 second sample pad, conjugate pad 6, and the control line 21 on T C 2 line 22 nitrocellulose membranes, aspirating pad 3 are sequentially disposed corresponding to the loading aperture 51, plus a liquid (reagent) hole 52, nitrocellulose membrane windows 23, window 31 aspirating pad. (如图3所示) (As shown in Figure 3)

[0067] 以下根据具体实施例对本发明作进一步说明: [0067] According to a particular embodiment the following Examples further illustrate the present invention:

[0068] 实施例一: [0068] Example a:

[0069] 人唾液绒毛膜促性腺激素(HCG)快速免疫诊断试纸的制备 Preparation of human saliva chorionic gonadotropin (HCG) rapid immunodiagnostic test strips [0069]

[0070] 1、所需原料如下: [0070] 1, the required starting material as follows:

[0071] 1.1试剂 [0071] 1.1 Reagents

[0072] 抗a-HCG和P-HCG抗体、纤维素纳米颗粒(Asahi Kasei公司产的纤维素纳米颗粒, 直径340±20mm)、酪蛋白、羟基磷灰石、海藻糖、NaCl、Na2HP04 • 12H20、NaH2P04 • 2H20、硼酸、硼砂、吐温-20、Triton X-100、鹿糖、超纯水等; [0072] a-HCG and anti-P-HCG antibody, nanoparticles cellulose (Asahi Kasei Corporation nanoparticles produced cellulose, diameter 340 ± 20mm), casein, hydroxyapatite, trehalose, NaCl, Na2HP04 • 12H20 , NaH2P04 • 2H20, boric acid, borax, Tween -20, Triton X-100, deer sugar, ultra pure water;

[0073] 1.2耗材 [0073] 1.2 consumables

[0074] 硝酸纤维素膜(NC膜)、8975玻璃纤维、GF-06玻璃纤维、吸水垫、底板4、容量瓶、锥形瓶、EP管、枪头; [0074] nitrocellulose membrane (NC film) 8975 glass fiber, GF-06 glass fibers, absorbent pads, a bottom plate 4, flasks, Erlenmeyer flasks, EP tube, pipette tip;

[0075] 1 • 3溶液配制 [0075] 1 • 3 solution preparation

[0076] PBS 缓冲液的配制:称取Na2HP04 • 12H20 1.615-2g,NaH2P04 • 2H20 0.225-5g, NaCl 4.0-5. Og于烧杯中,加入400ml超纯水溶解定容至500ml,0.22wii滤膜过滤后备用; [0077] 0.2M硼酸溶液的配制:称取硼酸12.37g于烧杯中,加入800ml超纯水溶解后定容至1L,0.22wii滤膜过滤后备用; [0076] PBS buffer is prepared: Weigh Na2HP04 • 12H20 1.615-2g, NaH2P04 • 2H20 0.225-5g, NaCl 4.0-5 Og beaker, 400ml of ultrapure water was added to dissolve volume to 500ml, 0.22wii membrane filtered standby; [0077] prepared 0.2M boric acid solution: Weigh 12.37g of boric acid in a beaker, 800ml of ultrapure water was added to dissolve the volume to 1L, the membrane filtration 0.22wii standby;

[0078] 0.05M硼砂溶液的配制:称取硼砂19.07g于烧杯中,加入800ml超纯水溶解后定容至IL,0.2 2wii滤膜过滤后备用; Formulation [0078] 0.05M borax solution: Weigh 19.07g borax beaker, 800ml of ultrapure water was added to dissolve the volume to IL, 0.2 2wii membrane filter after use;

[0079] IOmM pH8.0硼酸-硼砂缓冲液的配制:分别取3ml 0.0511硼砂溶液、7ml 0.211硼酸溶液于烧杯中,加入190ml超纯水,混匀备用; [0079] IOmM pH8.0 acid - borax buffer formulation: 3ml 0.0511 borax solution were taken, 7ml 0.211 boric acid solution in the beaker was added 190ml of ultrapure water and mix standby;

[0080] IOmM pH8.4硼酸-硼砂缓冲液的配制:分别取4.5ml 0.0511硼砂溶液、5.5ml 0.2M 硼酸溶液于烧杯中,加入190ml超纯水,混匀备用。 [0080] IOmM pH8.4 acid - borax buffer formulation: 4.5ml 0.0511 borax solution were taken, 5.5ml 0.2M boric acid solution in the beaker was added 190ml of ultrapure water and mix standby.

[0081] Sl、样本垫的制备 [0081] Preparation of Sl, the sample pad

[0082] 1.1、制备第一样本垫1 [0082] 1.1, the first sample pad prepared in 1

[0083] 先配制样本液:分别称取0.9g±0.05gNaCl、0.2g±0.05过氧化脲于100mL烧杯中, 加入70ml IOmM pH8.4硼酸盐缓冲液溶解,分别加入5ml马血清、Iml吐温-20及Iml Triton X-100,混勾,定容至100mL,0.22mi滤膜过滤后备用; [0083] The first sample was prepared: Weigh 0.9g ± 0.05gNaCl, 0.2g ± 0.05 carbamide peroxide in 100mL beaker, add 70ml IOmM pH8.4 borate buffer solution, were added to 5ml horse serum, discharge of Iml temperature -20 and Iml Triton X-100, mixing hook, the volume to 100mL, after membrane filtration 0.22mi standby;

[0084] 再将剪裁成0.7cm和3.2cm宽长条后的玻璃纤维膜置于上述配制的样本液中,室温浸泡120 ± 5分钟,37 °C烘干,即获得第一样本垫1,置于干燥皿中保存备用; [0084] and then cut into 0.7cm wide and 3.2cm long after the glass fiber membrane was placed in the above-described sample liquid formulation, soaking 120 ± 5 minutes at room temperature, 37 ° C dry, i.e., to obtain a first sample pad 1 , placed in a desiccator to save backup;

[0085] 1 • 2制备第二样本垫5 [0085] 1 • 2 sample pad prepared in a second 5

[0086] 选择大小尺寸符合要求,且孔经为500nm-1000um的玻璃纤维膜,然后在浓度为I Omg/ml的羟基磷灰石液体中室温浸泡120 ± 5分钟、37 °烘干、备用,且液体体积为200ml; [0087] S2、结合垫6的制备 [0086] sizes selected to meet the requirements, and for the wells of the 500nm-1000um glass fiber membrane, then immersed in a liquid the concentration of the hydroxyapatite I Omg / ml in 120 ± 5 minutes at room temperature, 37 ° drying, standby, and the liquid volume was 200ml; [0087] S2, bond pads 6 preparation of

[0088] 2.1、配制结合垫6处理液:称取0.2g± 0.05g酪蛋白,0.5g± 0.05g海藻糖于100mL 烧杯中,加入70ml IOmM pH8.0硼酸盐缓冲液溶解,加入0.3ml吐温-20,混匀,定容至100mL, 0 • 22wii滤膜过滤后备用; [0088] 2.1, formulated in conjunction with the treatment liquid pad 6: Weigh 0.2g ± 0.05g casein, 0.5g ± 0.05g trehalose in 100mL beaker, add 70ml IOmM pH8.0 borate buffer solution, was added 0.3ml Tween-20, mix, the volume to 100mL, 0 • 22wii membrane filter after use;

[0089] 2.2、纤维素纳米颗粒标记抗体:取5ml的离心管,依次加入20-30mM,9. OpH硼酸盐600-800ul及lmg/ml的P-HCG抗体500-600ul,再取已超声的0.1%纤维素纳米颗粒30〇111加入,混合均匀;37 °C孵育2h,每隔10分钟取出混匀片刻。 [0089] 2.2, the cellulose nanoparticle-labeled antibody: take 5ml centrifuge tube, sequentially adding 20-30mM, 9 OpH borate 600-800ul and lmg / ml of P-HCG antibody 500-600ul, ultrasound is then taken. 0.1% cellulose nanoparticles 30〇111 added, uniformly mixed; 37 ° C were incubated for 2h, 10 minutes, remove and mix every moment. 孵育完成后,向离心管中加2 %小鼠血清封闭液(或0.5 %纤维素纳米颗粒封闭液),均为6-8ml; 37°C封闭2h,每隔10分钟取出混合片刻,封闭结束后离心处理;14000g离心15-25min,尽量移除上清,沉淀用30-50mM,pH9-10.0硼酸盐溶解并恢复体积,混合均匀;沉淀用保存液、硼酸盐缓冲液,海藻糖各300ul溶解,置于4°C保存; After completion of the incubation, serum was added to the centrifuge tube mice 2% blocking solution (0.5% cellulose or nanoparticles blocking solution), were 6-8ml; 37 ° C closed 2h, 10 minutes, remove mixing every moment, a closed end after centrifugation; 14000 g centrifugation 15-25min, the supernatant was removed as far as possible, precipitated with 30-50mM, pH9-10.0 borate salt is dissolved and brought to volume, mix well; precipitated with preservation solution, borate buffer, each trehalose 300ul was dissolved, placed at 4 ° C;

[0090] 2.3、制备结合垫6:取结合垫6原料裁成0.7cm宽的长条后置于结合垫6处理液中室温浸泡4小时,37°C烘干,再用划膜喷金机将纤维素纳米颗粒标记抗体,按照12iiL/cm的量喷于结合垫6上,37度烘干后干燥皿中保存备用; [0090] 2.3 Preparation of bond pads 6: taken in conjunction with the rear gasket material 6 0.7cm wide strips cut bonding pad 6 is placed in the treatment liquid soaking temperature for 4 hours, 37 ° C dry, and then draw the film spraying machine cellulose nanoparticle-labeled antibody, in an amount 12iiL / cm is sprayed onto the bonding pad 6, after drying in a drying dish of 37 to save backup;

[0091] S3、硝酸纤维素膜2上检测T线21、质控C线22的包被 [0091] S3, the line 21 is detected on the T 2 nitrocellulose membrane, the C-line quality control package 22 is

[0092] 3.1、配制包被缓冲液:称取3g海藻糖于烧杯中,加入80-100mlPBS溶解后加入3- 5ml甲醇,混勾,定容至100ml,0.22wii滤膜过滤后备用; [0092] 3.1, coating formulation buffer: 3g trehalose was weighed in a beaker, was added 3- 5ml of methanol was added to dissolve the 80-100mlPBS, mixing hook, the volume to 100ml, 0.22wii membrane filtration after use;

[0093] 3.2、包被检测T线21:用移液枪取a-HCG抗体约IOOyL于EP管中,用包被缓冲液稀释至0.8-1.3mg/ml,按0.9-1.5yL/cm的量划在硝酸纤维素膜2的检测T线21上,37°C烘干后干燥皿中保存备用; [0093] 3.2, 21 packet line T is detected: a pipette with a-HCG antibody taken about IOOyL in EP tube, diluted with coating buffer to 0.8-1.3mg / ml, press 0.9-1.5yL / cm of detecting the amount of draw on the wire 2 T 21 nitrocellulose membrane, after drying 37 ° C were stored in a desiccator;

[0094] 3.3、包被质控C线22:用包被缓冲液将羊抗鼠多克隆抗体稀释至1.2-1.5mg/ml,按0.7-1.4yL/cm的量划在硝酸纤维素膜2的质控C线22上,37°C烘干后干燥皿中保存备用; [0095] S4、试纸的制备 [0094] 3.3, C-line quality control package is 22: The coating buffer with goat anti-mouse polyclonal antibody was diluted to 1.2-1.5mg / ml, according to the amount 0.7-1.4yL / cm nitrocellulose membrane in a scribe 2 22, 37 ° C after drying in a drying dish spare line C stored in the quality control; [0095] S4, preparation of test strips

[0096] 双通道试纸的制备:在常温的干燥室内,取底板4,先将包被的硝酸纤维素膜2贴合于底板4中部,将第一样本垫1与吸样垫3搭接于硝酸纤维素膜2两端上方,且第一样本垫1、 吸样垫3远离硝酸纤维素膜2的一端均与底板4平齐;第一样本垫1上方粘贴第二样本垫5,使得待测样本依次通过第二样本垫5、第一样本垫1、硝酸纤维素膜2、吸样垫3形成第一水平流动通道; [0096] Preparation of a dual-channel strip: the drying chamber at room temperature, taking the floor 4, first coated nitrocellulose membrane 2 bonded to the middle plate 4, the first absorbent sample pad and the loading pad 3 1 lap both ends of the above nitrocellulose membrane 2, and the first sample pad 1, pad aspirating end remote from the nitrocellulose membrane 3 2 4 are flush with the bottom plate; a first sample pad 1 attached over the second sample pad 5 , so that a second test sample passes through the sample pad 5, a first sample pad, a nitrocellulose membrane 2, the suction pad 3 is formed like a first horizontal flow channel;

[0097] 将纤维隔离薄膜7两端分别搭接在第一样本垫1、硝酸纤维素膜2上,结合垫6粘贴于纤维隔离薄膜7上方,且结合垫6-端与纤维隔离薄膜7的一侧平齐,另一端从纤维隔离薄膜7的另一侧延伸、搭接于硝酸纤维素膜2上,使得检测过程中所加的缓冲液体(试剂)依次通过结合垫6、纤维隔离薄膜7、硝酸纤维素膜2、吸样垫3形成第二水平流动通道,最后采用裁剪机剪切底板4后形成双通道试纸。 [0097] The fibrous insulation film 7 overlaps both ends at a first sample pad 1, 2, 6 bond pads attached to nitrocellulose membrane fibrous insulation film 7 above, 6- and 7 end with a fibrous insulation film bonding pads flush with the side, the other end extending from the other side of the fibrous insulation film 7, lap nitrocellulose film 2, so that the detection process of adding a buffer liquid (reagent) 6 passes through the conjugate pad, fibrous insulation film 7, a nitrocellulose membrane 2, the pad 3 is formed aspirating a second horizontal flow channels, Finally, the bottom plate cutting machine shear strip 4 forms a double passage. 对上述制备的试纸进行测试及结果判断 Of paper prepared as described above were tested and the results of determination

[0098] 将上述制备的试纸条放入待测液中停留5s,取出平放在台面上,当吸样垫3的颜色改变后(所述的第一水平流动通道),取50yLPBS缓冲液滴在结合垫6上,5min后读取结果(所述的第二水平流动通道),30min内有效。 [0098] The test strip into the test solution prepared above residence 5s, removed flat on the table, when the color change of the sample aspirating cushion 3 (the first flow passage level) taken 50yLPBS buffer 6 drops in the conjugate pad, reading result (the second horizontal flow channels) after 5min, 30min within valid.

[0099] 结果判断:当出现二条红色条带,一条位于检测T线21,另一条位于质控C线22,表明已怀孕;当出现一条红色条带,仅位于质控C线22,表明没有怀孕;当5-10分钟时在质控C 线22无红色条带出现,无论检测T线21有无红色条带出现,表明不正确的操作过程或者试纸条已变质损坏,需要用新的试纸条正确操作和/或重新测试。 [0099] Analyzing Results: appear when two red bands, one located at the detection line 21 T, and one in the C-line quality control 22, indicates that pregnant; when there is a red band, the C-line quality control is located only 22, showed no pregnancy; 5-10 minutes when the C band appears in the quality control line 22 no red bar, line T is detected regardless of presence or absence of 21 bands appear red, indicating an incorrect operation has been deteriorated or damaged strip, need new the correct operation of the test strip and / or re-testing.

[0100] 实施例二 [0100] Second Embodiment

[0101] 人唾液绒毛膜促性腺激素(HCG)快速免疫诊断试纸的灵敏度测试 [0101] human saliva chorionic gonadotropin (HCG) the sensitivity of rapid immunodiagnostic test strips

[0102] 1、唾液采集:用唾液擦拭法采集,采集唾液后放入唾液稀释液中,将采集棒两面分别在装有唾液稀释液的管壁上摩擦5-6次即可。 [0102] 1, Saliva collection: Saliva collection by swabbing, after collection of saliva into the saliva diluent, collected on both the sides of friction rod 5-6 times to the tube wall with saliva diluent.

[0103] 2、加标唾液:取98此唾液于微孔中,加入2.5yL m IU/ml的HCG标准品即得加标25mIU/ml的唾液;分别取98yL、95yL唾液于微孔中,加入2yL、5yL 100m IU/ml的HCG标准品即得加标2mIU/ml、5mIU/ml的唾液; [0103] 2, spiked saliva: Take 98 This saliva in micropores added 2.5yL m IU / ml of HCG standard that was spiked 25mIU ml saliva /; were taken 98yL, 95yL saliva in micropores, Add 2yL, 5yL 100m IU / ml of HCG standard that was spiked 2mIU / ml, 5mIU saliva / ml of;

[0104] 3、测试:将双通道试纸条平放桌面上,取50此唾液逐滴点在第二样本垫5上,室温静置l-5min,吸样垫3有颜色变化后,再取50yLPBS缓冲液逐滴点在结合垫6上,室温静置5min,观察结果,唾液灵敏度可达到2mIU/ml。 [0104] 3, test: two-channel strip flat on the table, this taking 50 points dropwise saliva sample pad 5 in a second, standing at room temperature l-5min, after aspirating pad 3 color change, and then take 50yLPBS buffer dropwise point bonding pad 6, allowed to stand at room temperature 5min, observations, saliva sensitivity can be achieved 2mIU / ml. 重复测试8次(如下表1)。 Test is repeated eight times (Table 1 below).

[0105] 表1人唾液HCG试纸条灵敏度测试 [0105] Table 1 Sensitivity in human saliva test strip HCG

[0106] [0106]

Figure CN106370871AD00111

[0107] 注:表1中,"+++,,表示明显可见,"++,,表示可见,"+,,表示微弱可见,"一,,表示不可见。 [0107] Note: In Table 1, "+++ indicates apparent ,," ,, represents visible ++ "indicates weak +, visible," a ,, for invisible. (C线表示质控线,T线表示检测线) (C line represents a control line, T line represents detection line)

[0108] 通过测试人唾液绒毛膜促性腺激素的快速免疫诊断试纸的灵敏度,空白唾液T线对照阴性,加标唾液(25mIU/ml)阳性明显可见,加标唾液(5mIU/ml)阳性大部分明显可见, 加标唾液(2mIU/ml)阳性可见,说明本试纸条灵敏度高。 [0108] the sensitivity rapid immunodiagnostic test strip hCG human saliva, saliva blank negative control line T, spiked saliva (25mIU / ml) is apparent positive spiked saliva (5mIU / ml) most positive apparent, spiked saliva (2mIU / ml) positive visible, indicating the high sensitivity of the present test strip.

[0109] 实施例三 [0109] Example three

[0110] 人唾液绒毛膜促性腺激素(HCG)试纸的37°C稳定性测试 [0110] human saliva chorionic gonadotropin (HCG) test paper 37 ° C stability test

[0111] 样品稀释液为1〇111]\1卬117.4?85,1%85厶,〇111111/11111^6标准品,25111111/11111^6标准品,从干燥器中取6支试纸条,备用;准备6份检测样品(Omiu/mlHCG标准品;25miu/ml HCG标准品),试纸条测试15min。 [0111] Liquid samples were diluted to 1〇111] \ 117.4 Ang 1? 85, 1 85% Si, 〇111111 / ^ 6 11111 standard, 25111111/11111 ^ 6 standards, taking the test strip 6 from the dryer standby; 6 parts by preparing a test sample (Omiu / mlHCG standard; 25miu / ml HCG standard), a test strip 15min. 测试结果如表2所示: The test results are shown in Table 2:

[0112] 表2人唾液HCG试纸条37 °C稳定性测试 [0112] Table 2 in human saliva test strip HCG 37 ° C stability test

[0113] [0113]

Figure CN106370871AD00112

[0114] 注:表2中,"+++"表示明显可见。 [0114] Note: In Table 2, "+++" represents visible. (C线表示质控线,T线表示检测线) (C line represents a control line, T line represents detection line)

[0115] 与编号1、2、3的对照组比较,结果显示信号正常,没有假阳性,试纸条稳定性良好。 [0115] Compared with the control group number of 2, 3, results show that the normal signal, no false positives, good stability of the test strip.

[0116] 实施例四 [0116] Fourth Embodiment

[0117] 人唾液绒毛膜促性腺激素(HCG)试纸的特异性测试 [0117] human saliva chorionic gonadotropin (HCG) strip of specific tests

[0118] 分别选取以下试剂: [0118] The following reagents were selected:

[0119] HCG,A液:0miu/ml HCG,10mM ?85^117.4,1%85八;肊6,8液:25111111/111111。6,1〇111]\1 PBS,pH7.4,l%BSA; [0119] HCG, A solution: 0miu / ml HCG, 10mM 85 ^ 117.4,1% 85 eight; Yi 6,8 liquid:? 25111111 / 111111.6,1〇111] \ 1 PBS, pH7.4, l% BSA;

[0120] hLH,A液:0miu/ml HCG,500miu/ml LH,10mM ?85^117.4,1%85厶;111^,8液:25111111/ ml HCG,500miu/ml LH,10mM PBS,pH7.4,l%BSA; [0120] hLH, A solution: 0miu / ml HCG, 500miu / ml LH, 10mM 85 ^ 117.4,1% 85 Si; 111 ^, 8 fluid:? 25111111 / ml HCG, 500miu / ml LH, 10mM PBS, pH7. 4, l% BSA;

[0121] hFSH,A液:0miu/ml HCG,1000miu/ml FSH,10mM ?85^117.4,1%85厶;1^511,8液: 25miu/ml HCG,1000miu/ml FSH,10mM PBS,pH7.4,l%BSA; [0121] hFSH, A solution: 0miu / ml HCG, 1000miu / ml FSH, 10mM 85 ^ 117.4,1% 85 Si; 1 ^ 511,8 liquid:? 25miu / ml HCG, 1000miu / ml FSH, 10mM PBS, pH7 .4, l% BSA;

[0122] hTSH,A液:0miu/ml HCG,1000uiu/ml TSH,10mM ?85^117.4,1%85八;11丁511,8液: 25miu/ml HCG,1000uiu/ml TSH,10mM PBS,pH7.4,l%BSA。 ? [0122] hTSH, A solution: 0miu / ml HCG, 1000uiu / ml TSH, 10mM 85 ^ 117.4,1% 85 eight; D 11 511,8 liquid: 25miu / ml HCG, 1000uiu / ml TSH, 10mM PBS, pH7 .4, l% BSA.

[0123] 从干燥器中取8支试纸条,备用。 [0123] The test strip 8 taken from the dryer, the standby. 准备4组AB液检测样品,试纸条测试15min(国家标准:测试A液T线不显色;测试B液T线显色。),测试结果如下表3、表4所示: AB Group 4 was prepared a test sample, the test strip 15min (National Standards: A test solution no color line T; T Test solution B the color line), the test results are as follows in Table 3, Table 4:

[0124] 表3人唾液HCG试纸条阴性特异性测试 [0124] Table 3 saliva HCG test strip specific test negative

[0125] [0125]

Figure CN106370871AD00121

[0126] 注:"+++"表示明显可见,"一"表示不可见。 [0126] Note: "+++" indicates visible, "a" indicates not visible.

[0127] 表4人唾液HCG试纸条阳性特异性测试 [0127] Table 4 human saliva HCG test strip specific test positive

[0128] [0128]

Figure CN106370871AD00122

[0129] 注:"+++"表示明显可见。 [0129] Note: "+++" indicates that visible.

[0130] (表3、4中,C线表示质控线,T线表示检测线) [0130] (Table 3,4, C line represents a control line, T line represents detection line)

[0131] 与编号1、2、3的HCG对照组比较,测试结果全部符合国标要求,特异性好,即本发明试纸条测试HCG与LH/FSH/TSH均无交叉。 [0131] Compared with the control group numbered 1,2,3 HCG, all test results meet the requirements of the national standard, specificity, i.e. HCG test strip of the present invention and LH / FSH / TSH no cross. 重复三次结果一致。 Repeat three times consistent results.

[0132] 实施例五 [0132] Embodiment V

[0133] 人唾液绒毛膜促性腺激素(HCG)试纸条的临床测试 [0133] human saliva chorionic gonadotropin (HCG) strip of clinical testing

[0134] 应用实施例一制备的人唾液绒毛膜促性腺激素(HCG)免疫层析试纸、进行医院临床测试,采用擦拭法收集临床唾液样本,按照本试纸条使用方法进行检测,以医院临床血液、尿检查结果进行对照,结果显示20份样本中有2份样本的血HCG值小于1.2mIU/ml,尿HCG 阴性,诊断为未怀孕,其它18份样本的血HCG值大于5mIU/ml,尿HCG阳性,诊断为怀孕,为阳性样本;将该20份唾液样本分别用本试纸条检测,试纸条检出的2份阴性样本,与医院临床血、尿结果一致且未出现假阳性,试纸条检出的其它18份样本均为阳性与医院临床结果一致且未出现假阴性,符合率100%。 A chorionic gonadotropin in human saliva was prepared in Example [0134] Applications of the embodiment (HCG) immunochromatographic strips, other hospital clinical testing, the collected saliva swabbing clinical samples for the detection method used according to the test strip, hospital clinical blood, urine test results were compared, the results show there are 20 parts 2 parts blood samples HCG sample is less than 1.2mIU / ml, urine HCG negative, diagnosis of non-pregnancy, HCG values ​​of other blood samples is greater than 18 parts by 5mIU / ml, urine HCG positive diagnosis of pregnancy, for the positive samples; the saliva samples were used 20 parts of the present detection strip, the strip negative samples detected two copies, hospitals and clinical blood, urine, and no false positive results are consistent , 18 test strip detection of other parts of the samples were positive and consistent clinical outcomes hospital and no false negative, in line with the rate of 100%. 试纸条临床测试见下表5: Clinical test strip Table 5 below:

[0135] 表5试纸条临床测试结果 [0135] Table 5 Test strip clinical results

[0136] [0136]

Figure CN106370871AD00131

[0137] [0137]

[0138] 注:表示不可见,"+++"表示明显可见、"++"表示可见"+"表示微弱可见。 [0138] Note: that is not visible, "+++" indicates that visible, "+" means visible "+" indicates a weak visible. (表5 中,C线表示质控线,T线表示检测线) (Table 5, C-line represents a control line, T line represents detection line)

[0139] 以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。 [0139] The foregoing is only preferred embodiments of the present invention, not intended to limit the present invention within the spirit and principle of the present invention, any modification, equivalent replacement, or improvement, it should be included in the present within the scope of the invention.

Claims (10)

1. 一种检测人唾液中绒毛膜促性腺激素的试纸,包括:第一样本垫(1)、包被有检测T线(21)与质控C线(22)的硝酸纤维素膜(2)、吸样垫(3)、底板(4),所述第一样本垫(1)、所述硝酸纤维素膜(2)、所述吸样垫⑶依次搭接于所述底板⑷,构成一检测试纸; 其特征在于:所述第一样本垫⑴上方粘贴一第二样本垫(5),用于过滤样本中杂质。 A saliva detection of human chorionic gonadotropin in the test paper, comprising: a first sample pad (1), coated with a nitrocellulose membrane detected with a T-line (21) and the quality control line C (22) ( 2), aspirating pad (3), the base plate (4), the first sample pad (1), the nitrocellulose membrane (2), said aspirating pad sequentially overlapped on the base plate ⑶ ⑷ constituting a test strip; wherein: the first sample and a second adhesive pad ⑴ above sample pad (5), for filtering impurities in the sample.
2. 根据权利要求1所述一种检测人唾液中绒毛膜促性腺激素的试纸,其特征在于:试纸还包括一两端均粘贴于所述硝酸纤维素膜(2)上的结合垫(6),且所述第一样本垫(1) 一端搭接于所述结合垫(6)上方,使得所述第一样本垫(1)、所述第二样本垫(5)、所述结合垫(6)、所述硝酸纤维素膜(2)、所述吸样垫(3)、所述底板(4)共同构成一单通道试纸。 The saliva for detecting human chorionic gonadotropin strip of claim 1, characterized in that: further comprising a strip attached at both ends of the cellulose nitrate film (2) on the bond pads (6 ), and the first sample pad (1) overlaps one end of said bond pads (6) above, such that the first sample pad (1), the second sample pad (5), the bond pads (6), the cellulose nitrate film (2), said aspirating pad (3), said base plate (4) together form a single channel strip.
3. 根据权利要求1所述一种检测人唾液中绒毛膜促性腺激素的试纸,其特征在于:试纸还包括上下依次层叠的结合垫(6)、纤维隔离薄膜(7),所述纤维隔离薄膜⑵两端分别搭接于所述第一样本垫(1)、所述硝酸纤维素膜⑵上方,且所述结合垫(6) -端与所述纤维隔离薄膜(7)的一侧平齐,另一端从所述纤维隔离薄膜(7)的另一侧延伸、搭接于所述硝酸纤维素膜⑵上,使得所述第一样本垫(1)、所述第二样本垫(5)、所述结合垫(6)、所述纤维隔离薄膜(7)、所述硝酸纤维素膜(2)、所述吸样垫(3)、所述底板(4)构成双通道试纸。 3. The saliva for detecting human chorionic gonadotropin in the strip in claim 1, wherein: the test strip further comprises upper and lower bound are stacked pad (6), fibrous insulation film (7), the fibrous insulation ⑵ overlapping film ends respectively to the first sample pad (1), above ⑵ the nitrocellulose membrane, and the bond pads (6) - one end of the fibrous insulation film (7) flush, and the other end extending from the other side of the fibrous insulation film (7), overlapping on nitrocellulose membrane is ⑵, such that the first sample pad (1), the second sample pad (5), said bond pads (6), the fibrous insulation film (7), the cellulose nitrate film (2), said aspirating pad (3), said base plate (4) a 2-channel strip .
4. 根据权利要求1所述一种检测人唾液中绒毛膜促性腺激素的试纸,其特征在于:所述第二样本垫(5)为500nm-lOOOum孔径的过滤试纸、或玻璃纤维膜、或无纺布或吸水滤纸,且所述第二样本垫(5)含有0. l-100mg/ml浓度的羟基磷灰石。 The saliva for detecting human chorionic gonadotropin strip of claim 1, wherein: said second sample pad (5) is 500nm-lOOOum pore size filter paper, glass fiber or film, or absorbent nonwoven fabric or filter paper, and the second sample pad (5) containing 0. l-100mg / ml concentration of hydroxyapatite.
5. 根据权利要求1所述一种检测人唾液中绒毛膜促性腺激素的试纸,其特征在于:所述第一样本垫⑴由玻璃纤维膜或无纺布构成;所述吸样垫⑶由吸水滤纸构成。 The saliva for detecting human chorionic gonadotropin strip of claim 1, wherein: the first sample is made of a glass fiber mat ⑴ film or a nonwoven fabric; the absorbent sample pad ⑶ composed of absorbent filter paper.
6. 根据权利要求1至5任意一项所述试纸的制备方法,其特征在于:包括如下步骤: S1:样本垫的制备第一样本垫(1)的制备:将剪裁后的玻璃纤维膜、或无纺布、或过滤试纸置于第一样本垫⑴处理液中室温浸泡、烘干、备用; 第二样本垫(5)的制备:选取孔径为500nm-1000um过滤试纸、或玻璃纤维膜、或无纺布、 或吸水滤纸,在浓度为0. ll〇〇mg/ml的羟基磷灰石液体中室温浸泡、烘干、备用; S2:结合垫(6)的制备:配制结合垫(6)处理液,标记抗体,将结合垫原料置于结合垫(6) 处理液中室温浸泡、烘干,再用划膜喷金机将纤维素纳米标记抗体或金标抗体或二者的混合物喷于经浸泡、烘干后的结合垫原料上、再烘干备用; S3:硝酸纤维素膜⑵包被配制包被缓冲液,用包被缓冲液分别稀释羊抗鼠多克隆抗体、抗α-HCG或β-HCG的抗体, 或者抗完整HCG抗体,获得质控C线(22)和检 The production method according to any one of the test strips of claims 1 to 5, characterized in that: comprising the steps of: Sl: preparing a first sample pad sample pad (1) Preparation of: glass fiber membrane cut or a nonwoven fabric, or filter paper placed in a first treatment liquid sample pad ⑴ immersed at room temperature, drying, standby; preparing a second sample pad (5): select a pore size of 500nm-1000um filter paper, glass fibers, or film, or a nonwoven fabric, or absorbent paper, at a concentration of 0.5 ll〇〇mg / ml of hydroxyapatite soaking liquid at room temperature, drying standby; S2: preparation of a pad (6) binding: formulating bond pads (6) the treatment liquid, a labeled antibody, the binding bond pads disposed feed pad (6) soaked in the treatment liquid at room temperature, dried, and then draw the film spraying machine cellulose nano gold-labeled antibody or labeled antibody or both the mixture was sprayed on immersion, pad binding material after drying, and then drying the standby; S3: ⑵ coated nitrocellulose membrane coating buffer formulated with coating buffer were diluted goat anti-mouse polyclonal antibody, anti- α-HCG or β-HCG antibody, or anti-HCG antibodies complete, the C-line quality control is obtained (22) and the subject T线(21); S4:试纸的制备单通道试纸的制备:在常温的干燥室内,取底板(4),先将包被的硝酸纤维素膜(2)贴合于底板⑷中部,将结合垫(6)粘贴于硝酸纤维素膜(2)上方,再将第一样本垫⑴一端搭接于结合垫(6)上方,另一端与底板(4) 一侧平齐;然后将吸样垫(3) -端搭接于硝酸纤维素膜(2)上方,另一端与底板(4)另一侧平齐后,将第二样本垫粘贴于第一样本垫(1)上方,最后采用裁剪机剪切底板⑷后形成试纸; 双通道试纸的制备:在常温的干燥室内,取底板(4),先将包被的硝酸纤维素膜(2)贴合于底板(4)中部,将第一样本垫(1)与吸样垫(3)搭接于硝酸纤维素膜(2)两端上方,且第一样本垫(1)、吸样垫⑶远离硝酸纤维素膜⑵的一端均与底板⑷平齐;第一样本垫⑴上方粘贴第二样本垫(5),使得待测样本依次通过第二样本垫(5)、第一样本垫(1)、硝酸纤维素膜(2)、吸样垫⑶形 T-line (21); S4: Preparation of a single-channel test strips of paper: room temperature in the drying chamber, to take the floor (4), first coated nitrocellulose membrane (2) is bonded to the central plate ⑷, binding the pad (6) attached to a nitrocellulose membrane (2) above, then the first lap end to a sample pad ⑴ bond pads (6) above, the other end of the base plate (4) is flush with the side; then aspirating pad (3) - overlapping ends nitrocellulose membrane (2) above, the other end of the base plate (4) is flush with the other side of the pad attached to the second sample of the first sample pad (1) above, and finally With cutter shear plate formed paper ⑷; preparation of a dual-channel strip: the drying chamber at room temperature, take the floor (4), the first packet of nitrocellulose membrane (2) bonded to the base plate (4) central, the first sample pad (1) and aspirating pad (3) overlapping a nitrocellulose membrane (2) over the ends, and the first sample pad (1), the sample absorbent pad remote from the nitrocellulose membrane ⑵ ⑶ are flush with the bottom end ⑷; ⑴ pasted over a first sample pad second sample pad (5), so that the test sample passes through the second sample pad (5), a first sample pad (1), nitrocellulose fibroin film (2), the sample absorbent pad shaped ⑶ 成第一水平流动通道; 将纤维隔离薄膜(7)两端分别搭接在第一样本垫(1)、硝酸纤维素膜(2)上,结合垫(6) 粘贴于纤维隔离薄膜⑴上方,且结合垫(6) -端与纤维隔离薄膜⑵的一侧平齐,另一端从纤维隔离薄膜(7)的另一侧延伸、搭接于硝酸纤维素膜(2)上,使得检测过程中所加的缓冲液体依次通过结合垫(6)、纤维隔离薄膜(7)、硝酸纤维素膜(2)、吸样垫⑶形成第二水平流动通道,最后采用裁剪机剪切底板⑷后形成双通道试纸。 A first horizontal flow channel; the fibrous insulation film (7) overlaps the two ends of the first sample pad (1), nitrocellulose membrane (2), bond pads (6) bonded to the upper fibrous insulation film ⑴ and the bond pads (6) - side end flush ⑵ fibrous insulation film, and the other end extending from the other side of the fibrous insulation film (7), lapping nitrocellulose membrane (2), such that the detector after the increase of the damping fluid passes through the conjugate pad (6), fibrous insulation film (7), a nitrocellulose membrane (2), aspirating a second pad formed ⑶ horizontal flow channels, Finally shear cutter plate formed ⑷ dual-channel strip.
7. 根据权利要求6所述制备方法,其特征在于: 步骤S1中,所述第一样本垫(1)、所述第二样本垫⑶制备过程中的浸泡时间均为120土511^11,烘干温度均为37°。 The preparation method according to claim 6, wherein: the step S1, the first sample pad (1), the second soak time Sample preparation ⑶ pad 120 are in the soil 11 ^ 511 drying temperatures are 37 °.
8. 根据权利要求6所述制备方法,其特征在于: 步骤S1中,第一样本垫⑴处理液的配制方法为:称取0.5g± 0.05g BSA于烧杯中,加入70ml 10mM pH8.4硼酸盐缓冲液溶解,加入吐温-20,混匀,定容、滤膜过滤后备用。 8. The production method according to claim 6, wherein: the step S1, a first sample pad ⑴ preparation method for the treatment liquid: Weigh 0.5g ± 0.05g BSA beaker, was added 70ml 10mM pH8.4 borate buffer solution, Tween-20 was added, mixed, volume, membrane filtration after use.
9. 根据权利要求6所述制备方法,其特征在于: 步骤S2中,结合垫(6)处理液的制备方法:称取0.2g ± 0.05g酪蛋白、0.5g ± 0.05g海藻糖于烧杯中,加入70ml 10mM pH8.0硼酸盐缓冲液溶解,加入吐温-20,混匀、滤膜过滤后备用; 且制得的所述结合垫(6)上有150-550nm纤维素纳米颗粒,10-80nm胶体金或者二者的混合物,且标记有HCG抗体α、β特异性抗体或者抗完整HCG抗体;或有乳胶颗粒或碳颗粒或其它发光染料或酶,且结合有HCG抗体α、β特异性抗体或者抗完整HCG抗体,或引用或结合亲和素、生物素。 9. The production method according to claim 6, wherein: the step S2, the bonding pads preparation (6) Processing solution: Weigh 0.2g ± 0.05g casein, 0.5g ± 0.05g trehalose beaker adding 70ml 10mM pH8.0 borate buffer solution, Tween-20 was added, mixed, membrane filtration after use; and incorporating the obtained nano particles 150-550nm cellulose pad (6), 10-80nm colloidal gold or a mixture of both, and HCG antibody labeled with α, β-specific antibodies or an anti-HCG antibody complete; or latex particles or carbon particles, or other enzymes or luminescent dyes, and HCG antibodies bound α, β specific antibodies or an anti-HCG antibody complete, or reference, or binding affinity of biotin.
10. 根据权利要求6所述制备方法,其特征在于: 步骤S3中,包被缓冲液的制备方法为:取海藻糖于烧杯中,加入PBS溶解后再加入甲醇, 混匀、定容、滤膜过滤后备用; 且所述硝酸纤维素膜⑵的检测Τ线上(21)设置有亲和素、生物素。 10. The preparation method according to claim 6, wherein: the step S3, preparation of coating buffer: taking Trehalose in a beaker, dissolved in PBS was added and then methanol is added, mix, constant volume, filter after filtration membrane standby; ⑵ nitrocellulose membranes and the detection Τ line (21) is provided with avidin, biotin.
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