CN106248975A

CN106248975A - A kind of H HCG Rapid immunodiagnosis chromatographic test paper and preparation method thereof

Info

Publication number: CN106248975A
Application number: CN201610541351.2A
Authority: CN
Inventors: 马北峰; 马北阳; 徐晓峰; 谢从平; 李娟娟
Original assignee: Wuhan Hundred Biological Technology Co Ltd
Current assignee: Wuhan Hundred Biological Technology Co Ltd
Priority date: 2016-07-11
Filing date: 2016-07-11
Publication date: 2016-12-21

Abstract

A kind of H HCG Rapid immunodiagnosis chromatographic test paper, it is characterized in that: include the sample pad being sequentially connected with, be coated with detection T line and the nitrocellulose filter of Quality Control C line, sample suction pad, and described sample pad, described nitrocellulose filter, described sample suction pad are all pasted onto in a gripper shoe.Its advantage is: 1., be coated H HCG specific antibody B207 or B152 on pad, nitrocellulose filter, or anti alpha HCG or the antibody of β HCG, or by embody such as blood, urine, the detection of saliva equal samples of human body H HCG content, the design of anti-complete HCG antibody, can know that the most conceived or anemia of pregnant woman the placental function of detected person is the best and predicts miscarriage etc.；2., this care diagnostic reagent utilizing immune chromatography test paper method quickly to detect, it is suitable for normal pregnancy and the diagnosis and differential diagnosis of abnormal gestation in early days, there is high specificity, sensitivity high, have safety, easily preserve, good stability and the advantage such as application is convenient.

Description

H-HCG rapid immunodiagnosis chromatography test paper and preparation method thereof

Technical Field

The invention relates to the technical field of medical detection, in particular to a H-HCG rapid immunodiagnosis chromatography test paper and a preparation method thereof.

Background

Human Chorionic Gonadotropin (HCG) is a glycoprotein hormone secreted by placental trophoblast cells and having a molecular weight of 36700, and is composed of alpha and beta subunits. HCG is the most commonly used marker in clinical pregnancy and pregnancy related disease examinations, and its bioimmunological characteristics are mainly determined by the β subunit, and in order to avoid cross-reactivity of anti-HCG antibodies with other polypeptide hormones such as FSH, LH and TSH, although intact HCG is found in the most common form during pregnancy, HCG exists in different forms in blood, urine, and other subtypes or fragments of HCG are also present in the blood, urine and saliva of pregnant women, including highly glycosylated human chorionic gonadotropin (H-HCG), free HCG- α units, free HCG- β units, HCG- β core fragments and gap HCG, which have different biological functions, respectively. However, the detection tools currently in widespread use for detecting human HCG are HCG colloidal gold immunochromatographic test strips, and methods for detecting total HCG concentration in pregnant woman serum using anti-HCG α and β chain or β -HCG monoclonal antibodies, all of which have HCG values less than the actual sum of the various HCG components, and do not fully bind all types or subtypes or fragments of HCG, nor do they provide information on the various subtypes, particularly their biological function and related clinical significance.

Early pregnancy can be divided into normal pregnancy and abnormal pregnancy, the abnormal pregnancy comprises ectopic pregnancy and spontaneous abortion, the morbidity is about 1% and 10-15% respectively in China, and the early pregnancy is in a trend of rising year by year, is one of main death reasons of pregnant and lying-in women, and has become an important health problem for women of childbearing age. Although we have conducted quantitative measurements of progesterone and HCG levels in pregnant women for the determination of ectopic pregnancy and spontaneous abortion, no screening method has been available to date for rapid, accurate and immediate diagnosis. There is also no separate rapid, accurate instant diagnostic method to distinguish between early normal pregnancy and abnormal pregnancy. Currently, the method of clinical use is one of ultrasound examination and the other is the HCG doubling test to determine the presence of ectopic pregnancy. The existence of the embryo is judged by judging whether the embryo sac exists or not through vaginal ultrasound, and the existence of the ectopic pregnancy is judged through abdominal ultrasound. However, the method for detecting the embryo and the embryo sac in the early stage by adopting the ultrasound is suitable for 5-8 weeks of pregnancy (about 1-4 weeks after menopause), the diagnosis of pregnant women with ectopic pregnancy symptoms often misses the time of the ultrasound detection, and the diagnosis is carried out by relying on an HCG doubling test, if the HCG level in the blood of the pregnant women is not doubled within two days, the ectopic pregnancy is indicated. On the contrary, the person who has suffered spontaneous abortion cannot adopt the above two analysis methods. Measuring the doubling rate of HCG every two days has become a standard gynecological examination, and decreased levels and non-doubling of HCG over two days often indicate pregnancy failure. However, the method has the defects that a pregnant woman needs to be treated twice, a pregnant woman needs to draw blood twice to detect the HCG level more than twice, the detection results of the three are limited, the sensitivity of the detection is 62-78% through a large number of different literature reports, and the false positive rate is as high as 26-40%.

The detection of the level of hyperglycosylated human chorionic gonadotropin (H-HCG) can well monitor or distinguish early pregnancy as normal pregnancy and abnormal pregnancy, because H-HCG is a hormone normally secreted in early pregnancy and has the function of completing implantation of a placenta into an endometrium, so that for normal pregnancy, insufficient secretion of H-HCG can cause implantation and growth disorder of the placenta, cause placental dysplasia and cause abortion. Research shows that the efficiency of predicting abortion by serum H-HCG at 4-7 weeks of gestation (according to the last menstruation) is higher than that of conventional HCG examination. Sasaki et al detected the ratio of H-HCG to total HCG at about 7 days of gestation (based on LH-peak) to predict abortion and obtained 100% positive predictive value and 75% negative predictive value. Another study on In Vitro Fertilization (IVF) pregnant women showed that H-HCG testing 3 weeks after embryo implantation predicted pregnancy outcome better than routine HCG testing after IVF and distinguished whether the cause of abortion was embryo implantation failure or clinical spontaneous abortion, and that the testing of serum and uterine tissue on day 6 after IVF surgery by Strom et al also found that H-HCG had 100% sensitivity and specificity for the diagnosis of successful pregnancy. And can identify biochemical pregnancy. Cole found in recent studies that there was not much difference in serum HCG concentration between the successful pregnancy and the spontaneous abortion, but the H-HCG concentration in the serum of women with spontaneous abortion was significantly lower than that of the successful pregnancy, while the H-HCG level in the serum of biochemical pregnancy was lower. The detection of H-HCG has superiority and important clinical significance in the aspects of early pregnancy, ectopic pregnancy, early abortion, tumor monitoring and the like. Therefore, it is urgent to research how to apply H-HCG to products to prepare products with fast detection speed and convenience.

Disclosure of Invention

The invention aims to solve the technical defects and provides the H-HCG rapid immunodiagnosis chromatography test paper and the preparation method thereof, the method is convenient, rapid and high in accuracy, the test paper is suitable for diagnosis and differential diagnosis of early normal pregnancy and abnormal pregnancy, and has important clinical significance in the aspects of early pregnancy, ectopic pregnancy, early abortion, tumor monitoring and the like.

In one aspect of the present invention, there is provided a rapid immunodiagnosis test strip for H-HCG, comprising: the kit comprises a sample pad, a nitrocellulose membrane coated with a detection T line and a quality control C line and a sample sucking pad which are sequentially connected, wherein the sample pad, the nitrocellulose membrane and the sample sucking pad are sequentially overlapped and stuck on a supporting plate;

preferably, the H-HCG rapid immunodiagnosis test paper further comprises a combination pad, and the combination pad is arranged between the sample pad and the nitrocellulose membrane, so that the sample pad, the combination pad, the nitrocellulose membrane and the sample sucking pad form a single-channel test paper;

or

The H-HCG rapid immunodiagnosis test paper further comprises a fiber isolation film, a combination pad and a liquid adding pad, wherein two ends of the fiber isolation film are respectively lapped on the sample pad and above the nitrocellulose membrane, two ends of the combination pad are respectively lapped on the fiber isolation film and above the nitrocellulose membrane, and two ends of the liquid adding pad are respectively lapped on the fiber isolation film and above the combination pad, so that the sample pad, the fiber isolation film, the combination pad, the liquid adding pad, the nitrocellulose membrane and the sample sucking pad form a dual-channel test paper.

Further, the air conditioner is provided with a fan,

an H-HCG specific antibody B207 or B152 is arranged on the bonding pad, the detection T line on the nitrocellulose membrane is coated with the H-HCG specific antibody B152 or B207, and the quality control C line contains a goat anti-mouse polyclonal antibody;

or,

an anti-alpha-HCG or anti-beta-HCG antibody, or an anti-complete HCG antibody, or an H-HCG specific antibody B207 or B152 is arranged on the combination pad; the detection T line consists of a detection line T1 and a detection line T2, the detection line T1 is coated with an antibody resisting alpha-HCG or beta-HCG, or an antibody resisting complete HCG, the detection line T2 is coated with an H-HCG specific antibody B152 or B207, or the detection line T1 is coated with an H-HCG specific antibody B152 or B207, and the detection line T2 is coated with an antibody resisting alpha-HCG or beta-HCG, or an antibody resisting complete HCG; suitable for an immune competition method, the nitrocellulose membrane (3) is coated with H-HCG antigen on a T line or vice versa, the binding pad (2) is provided with the H-HCG antigen, and the T line is coated with H-HCG specific antibody.

In a second aspect of the present invention, there is provided a method for preparing the H-HCG rapid immunodiagnostic chromatographic test strip, comprising the steps of:

preparing a sample pad: preparing a sample pad treatment solution, and then placing the cut glass fiber film or non-woven fabric in the sample pad treatment solution for soaking at room temperature and drying for later use;

preparing a bonding pad: preparing a bonding pad treatment solution and a gold-labeled antibody, placing the bonding pad raw material in the bonding pad treatment solution for soaking at room temperature and drying, spraying the gold-labeled antibody on the soaked bonding pad raw material by using a film-scratching gold-spraying machine, and drying for later use;

③ coating the nitrocellulose membrane: preparing a coating buffer solution, and respectively diluting a goat anti-mouse polyclonal antibody, an anti-alpha-HCG or beta-HCG antibody, or an anti-complete HCG antibody (or the antibodies can be monoclonal antibodies, polyclonal antibodies or various fragments of the antibodies), and an H-HCG monoclonal antibody B207 or B152 to 0.1-20mg/ml by using the coating buffer solution to obtain a quality control C line, a detection line T1, a detection line T2 or a quality control C line, a detection line T2, a detection line T1 or a quality control C line and a detection T line;

fourthly, preparing the test paper:

single-channel test paper: in a drying chamber at normal temperature, taking the supporting plate, firstly attaching the coated nitrocellulose membrane to the middle part of the supporting plate, then respectively attaching the combination pad and the sample sucking pad to two sides of the nitrocellulose membrane, attaching the sample pad to one side of the combination pad opposite to the nitrocellulose membrane, and finally cutting the supporting plate by adopting a cutting machine;

double-channel test paper: the sample sucking pad is adhered to one side of the nitrocellulose membrane, one side of the sample sucking pad is in lap joint with the nitrocellulose membrane, the other side edge of the sample sucking pad is flush with the support plate, and the other side of the nitrocellulose membrane is in lap joint with the sample pad, so that a sample to be detected sequentially passes through the sample pad, the nitrocellulose membrane and the sample sucking pad to form a first horizontal flow channel;

will fibre barrier film both ends overlap joint respectively the sample pad cellulose nitrate membrane top, the joint pad both ends overlap joint respectively the fibre barrier film cellulose nitrate membrane top, liquid feeding pad both ends overlap joint respectively the fibre barrier film the joint pad top for buffer solution loops through in the testing process the liquid feeding pad the joint pad cellulose nitrate membrane inhale the appearance and fill up and form the horizontal flow channel of second, adopt the cutter to cut at last the backup pad.

Preferably, in the step (i), the preparation method of the sample treatment solution includes: weighing 0.5g +/-0.05 g BSA in a beaker, adding 70ml 10mM borate buffer solution with pH8.4 for dissolving, adding Tween-20, mixing uniformly, fixing the volume, and filtering with a filter membrane for later use.

Preferably, in the second step, the preparation method of the bonding pad treating solution comprises: weighing 0.1g +/-0.05 g BSA and 0.5g +/-0.05 g trehalose in a beaker, adding 70ml 10mM borate buffer solution with pH8.0 for dissolving, adding Tween-20, uniformly mixing, and filtering with a filter membrane for later use; the gold-labeled antibody contains an anti-alpha-HCG or anti-beta-HCG antibody, or an anti-complete HCG antibody, or an H-HCG specific antibody B207 or B152.

In the third step, the preparation method of the coating buffer solution comprises the following steps: dissolving trehalose in PBS, adding methanol, mixing, diluting to desired volume, and filtering with filter membrane.

Further, the sample pad material is a glass fiber film or a non-woven fabric; the sample absorbing pad is composed of water absorbing filter paper.

Further, the binding pad (2) is composed of 5-100nm colloidal gold or 5-800nm nano-material or latex particles or carbon particles or other luminescent dyes or enzyme-bound anti-HCG antibody or H-HCG antibody, or quoted or bound with avidin and biotin; the binding pad (2) is added with avidin or biotin in colloidal gold or cellulose nanoparticles labeled with specific antibodies, or colloidal gold-cellulose nanoparticles labeled with specific antibodies, or both.

The invention relates to a H-HCG rapid immunodiagnosis chromatography test paper and a preparation method thereof, and the test paper has the advantages that:

firstly, a binding pad and a nitrocellulose membrane are coated with an H-HCG (human chorionic gonadotropin) specific antibody B207 or B152, or an anti-alpha-HCG or anti-beta-HCG antibody, or an anti-complete HCG antibody, so that whether a detected person is pregnant or not, or whether the placenta function of a pregnant woman is good or not, prediction of abortion and the like can be obtained through detection of samples such as blood, urine, saliva and the like of human body H-HCG content; the H-HCG test paper is suitable for the early pregnancy, particularly the detection can be started 5-7 days after the same house or 1-2 days before the expiration day of the menstruation, and the H-HCG test paper can be used for preliminary diagnosis of early pregnancy women by all levels of medical institutions and self detection of the women at home;

the diagnostic reagent for rapid detection by utilizing the immunochromatographic test paper method is particularly suitable for diagnosis and differential diagnosis of early normal pregnancy and abnormal pregnancy, has the advantages of strong specificity, high sensitivity, safety, easy storage, good stability, convenient application and the like, and has great application value and clinical significance in the aspects of medical rapid diagnosis, health physical examination, general survey and family diagnosis, and medical health and prenatal and postnatal care of women.

Drawings

FIG. 1 and FIG. 2 are schematic diagrams of a single-channel structure of an H-HCG rapid immunodiagnosis chromatography test paper;

FIG. 3 and FIG. 4 are schematic diagrams of a double-channel structure of an H-HCG rapid immunodiagnosis chromatography test paper;

FIG. 5 is a schematic view of a two-pass outer shell construction;

wherein:

1. sample pad, 2, combination pad, 21, liquid adding pad, 3, nitrocellulose membrane, 31, detection T line, 32, detection lines T1, 33, detection lines T2, 34, quality control C line, 4, sample sucking pad, 5, support plate, 6, fiber isolation film, 7, sample adding hole, 8, liquid adding hole, 9, nitrocellulose membrane window, 10 and sample sucking pad window.

Detailed Description

The utility model provides a quick immunodiagnosis chromatography test paper of H-HCG, includes sample pad 1, the cladding have detect T line 31 and quality control C line 34 nitrocellulose membrane 3, inhale a kind pad 4 that connect gradually, just sample pad 1 nitrocellulose membrane 3 inhale a kind pad 4 overlap joint in proper order, paste on a backup pad 5.

Preferably, the H-HCG rapid immunodiagnostic chromatographic test strip: the test strip further comprises a combination pad 2, and the combination pad 2 is arranged between the sample pad 1 and the nitrocellulose membrane 3, so that the sample pad 1, the combination pad 2, the nitrocellulose membrane 3, and the sample absorbing pad 4 form a single-channel test strip (as shown in fig. 1 and fig. 2).

Preferably, the H-HCG rapid immunodiagnostic chromatographic test strip: the test paper also comprises a fiber isolation film 6, a combination pad 2 and a liquid adding pad 21, wherein two ends of the fiber isolation film 6 are respectively lapped above the sample pad 1 and the nitrocellulose membrane 3, two ends of the combination pad 2 are respectively lapped above the fiber isolation film 6 and the nitrocellulose membrane 3, and two ends of the liquid adding pad 21 are respectively lapped above the fiber isolation film 6 and the combination pad 2, so that the sample pad 1, the fiber isolation film 6, the combination pad 2, the liquid adding pad 21, the nitrocellulose membrane 3 and the sample sucking pad 4 form a double-channel test paper (shown in figures 3 and 4);

further, the air conditioner is provided with a fan,

an H-HCG specific antibody B207 or B152 is arranged on the binding pad 2, the H-HCG specific antibody B152 or B207 is coated on the nitrocellulose membrane 3, and the quality control C line 34 contains a goat anti-mouse polyclonal antibody;

or,

an anti-alpha-HCG or anti-beta-HCG antibody, or an anti-complete HCG antibody, or an H-HCG specific antibody B207 or B152 is arranged on the binding pad 2; the detection T line 31 consists of a detection line T132 and a detection line T233, wherein the detection line T132 is coated with an antibody against alpha-HCG or beta-HCG, or an antibody against complete HCG, the detection line T233 is coated with an antibody B152 or B207 specific to H-HCG, or the detection line T132 is coated with an antibody B152 or B207 specific to H-HCG, and the detection line T233 is coated with an antibody against alpha-HCG or beta-HCG, or an antibody against complete HCG;

or,

suitable for an immune competition method, the nitrocellulose membrane 3T line is coated with H-HCG antigen or vice versa, the binding pad 2 is provided with H-HCG antigen, and the T line is coated with H-HCG specific antibody.

Preferably, the sample pad 1 material is a glass fiber film or a non-woven fabric; the sample absorbing pad 4 is made of water absorbing filter paper.

Preferably, the binding pad 2 is composed of 5-100nm colloidal gold or 5-800nm nano-material or latex particles or carbon particles or other luminescent dye or enzyme bound anti-HCG antibody or H-HCG antibody, or quoted or bound avidin, biotin; the binding pad 2 is added with avidin or biotin, or is added with colloidal gold or cellulose nanoparticles labeled with specific antibodies, or colloidal gold-cellulose nanoparticles labeled with specific antibodies, or both.

The detection T line 31 is provided with an immobilized antibody or H-HCG antigen or added with biotin or avidin.

In the above, the H-HCG specific antibodies B207 and B152 are used in an amount of 0.01 to 100mg/ml, and the anti- α -HCG or β -HCG antibody, or the anti-whole HCG antibody is used in an amount of 0.01 to 100 mg/ml.

The invention relates to a H-HCG rapid immunodiagnosis chromatography test paper, which has the following specific form:

firstly, the combination pad 2 contains an H-HCG specific antibody B207, the detection T line 31 is a nitrocellulose membrane 3 coated with an H-HCG specific antibody B152, and the quality control C line 34 is a goat anti-mouse polyclonal antibody coated on the nitrocellulose membrane 3;

secondly, the combination pad 2 contains an H-HCG specific antibody B207, the detection T line 31 is a nitrocellulose membrane 3 coated with the H-HCG specific antibody B207, and the quality control C line 34 is a goat anti-mouse polyclonal antibody coated on the nitrocellulose membrane 3;

thirdly, the combination pad 2 contains an H-HCG specific antibody B152, the detection T line 31 is a nitrocellulose membrane 3 coated with the H-HCG specific antibody B207, and the quality control C line 34 is a goat anti-mouse polyclonal antibody coated on the nitrocellulose membrane 3;

fourthly, the combination pad 2 contains an H-HCG specific antibody B152, the detection T line 31 is formed by coating the H-HCG specific antibody B152 on the nitrocellulose membrane 3, and the quality control C line 34 is formed by coating a goat anti-mouse polyclonal antibody on the nitrocellulose membrane 3;

fifthly, the combination pad 2 contains an H-HCG specific antibody B207, a detection line T132 is formed by coating an anti-alpha-HCG antibody on a nitrocellulose membrane 3, a detection line T233 is formed by coating an H-HCG specific antibody B152 on the nitrocellulose membrane 3, and a quality control C line 34 is a goat anti-mouse polyclonal antibody coated on the nitrocellulose membrane 3;

sixthly, the combination pad 2 contains an H-HCG specific antibody B207, a detection line T132 is formed by coating an anti-beta-HCG antibody on a nitrocellulose membrane 3, a detection line T233 is formed by coating an H-HCG specific antibody B152 on the nitrocellulose membrane 3, and a quality control C line 34 is a goat anti-mouse polyclonal antibody coated on the nitrocellulose membrane 3;

seventhly, the combination pad 2 contains an H-HCG specific antibody B207, the detection line T132 is formed by coating an H-HCG specific antibody B152 on the nitrocellulose membrane 3, the detection line T233 is formed by coating an anti-beta-HCG antibody on the nitrocellulose membrane 3, and the quality control C line 34 is a goat anti-mouse polyclonal antibody coated on the nitrocellulose membrane 3;

eighthly, the combination pad 2 contains an H-HCG specific antibody B207, the detection line T132 is formed by coating an H-HCG specific antibody B152 on the nitrocellulose membrane 3, the detection line T233 is formed by coating an anti-alpha-HCG antibody on the nitrocellulose membrane 3, and the quality control C line 34 is a goat anti-mouse polyclonal antibody coated on the nitrocellulose membrane 3;

ninthly, the combination pad 2 contains an H-HCG specific antibody B207, a detection line T132 is formed by coating an anti-alpha-HCG antibody on a nitrocellulose membrane 3, a detection line T233 is formed by coating an anti-beta-HCG antibody on the nitrocellulose membrane 3, and a quality control C line 34 is formed by coating a goat anti-mouse polyclonal antibody on the nitrocellulose membrane 3;

tenthly, the combination pad 2 contains an H-HCG specific antibody B207, a detection line T132 is formed by coating an anti-beta-HCG antibody on a nitrocellulose membrane 3, a detection line T233 is formed by coating an anti-alpha-HCG antibody on the nitrocellulose membrane 3, and a quality control C line 34 is formed by coating a goat anti-mouse polyclonal antibody on the nitrocellulose membrane 3;

eleventh, the combination pad 2 contains an H-HCG specific antibody B152, the detection line T132 is a nitrocellulose membrane 3 coated with an H-HCG specific antibody B207, the detection line T233 is a nitrocellulose membrane 3 coated with an anti-beta-HCG antibody, and the quality control C line 34 is a goat anti-mouse polyclonal antibody coated on the nitrocellulose membrane 3;

twelve, the combination pad 2 contains H-HCG specific antibody B152, the detection line T132 is the cellulose nitrate membrane 3 coated with anti-beta-HCG antibody, the detection line T233 is the cellulose nitrate membrane 3 coated with H-HCG specific antibody B207, the quality control C line 34 is the goat anti-mouse polyclonal antibody coated on the cellulose nitrate membrane 3;

thirteen, the combination pad 2 is provided with an H-HCG specific antibody B152, the detection line T132 is a nitrocellulose membrane 3 coated with an anti-alpha-HCG antibody, the detection line T233 is a nitrocellulose membrane 3 coated with an HCG specific antibody B207, and the quality control C line 34 is a goat antibody coated on the nitrocellulose membrane 3 and used for resisting mouse polyclonal antibody;

fourteen, the combination pad 2 is provided with an H-HCG specific antibody B152, the detection line T132 is formed by coating an H-HCG specific antibody B207 on the nitrocellulose membrane 3, the detection line T233 is formed by coating an anti-alpha-HCG antibody on the nitrocellulose membrane 3, and the quality control C line 34 is a goat antibody which is coated on the nitrocellulose membrane 3 and resists mouse polyclonal;

fifteen, the combination pad 2 is provided with an H-HCG specific antibody B152, the detection line T132 is a nitrocellulose membrane 3 coated with an anti-alpha-HCG antibody, the detection line T233 is a nitrocellulose membrane 3 coated with an anti-beta-HCG antibody, and the quality control C line 34 is a goat antibody coated on the nitrocellulose membrane 3 and used for resisting mouse polyclonal antibody;

sixthly, the binding pad 2 is provided with an H-HCG specific antibody B152, the detection line T132 is formed by coating an anti-beta-HCG antibody on the nitrocellulose membrane 3, the detection line T233 is formed by coating an anti-alpha-HCG antibody on the nitrocellulose membrane 3, and the quality control C line 34 is a goat antibody which is coated on the nitrocellulose membrane 3 and is used for resisting mouse polyclonal;

seventhly, the combination pad 2 is provided with an anti-alpha-HCG antibody, a detection line T132 is formed by coating an H-HCG specific antibody B207 on the nitrocellulose membrane 3, a detection line T233 is formed by coating an H-HCG specific antibody B152 on the nitrocellulose membrane 3, and a quality control C line 34 is formed by coating a goat antibody of anti-mouse polyclonal on the nitrocellulose membrane 3;

eighteen, the combination pad 2 is provided with an anti-alpha-HCG antibody, the detection line T132 is formed by coating an H-HCG specific antibody B152 on the nitrocellulose membrane 3, the detection line T233 is formed by coating an H-HCG specific antibody B207 on the nitrocellulose membrane 3, and the quality control C line 34 is a goat antibody which is coated on the nitrocellulose membrane 3 and is used for resisting mouse polyclonal;

nineteen, the combination pad 2 is provided with an anti-alpha-HCG antibody, the detection line T132 is formed by coating an anti-beta-HCG antibody on the nitrocellulose membrane 3, the detection line T233 is formed by coating an H-HCG specific antibody B207 on the nitrocellulose membrane 3, and the quality control C line 34 is a goat antibody which is coated on the nitrocellulose membrane 3 and resists mouse polyclonal;

twenty, the combination pad 2 is provided with an anti-alpha-HCG antibody, the detection line T132 is formed by coating an anti-beta-HCG antibody on the nitrocellulose membrane 3, the detection line T233 is formed by coating an H-HCG specific antibody B152 on the nitrocellulose membrane 3, and the quality control C line 34 is a goat antibody which is coated on the nitrocellulose membrane 3 and is used for resisting mouse polyclonal;

twenty one, the combination pad 2 is provided with an anti-alpha-HCG antibody, the detection line T132 is formed by coating an H-HCG specific antibody B207 on the nitrocellulose membrane 3, the detection line T233 is formed by coating an anti-beta-HCG antibody on the nitrocellulose membrane 3, and the quality control C line 34 is a goat antibody which is coated on the nitrocellulose membrane 3 and resists mouse polyclonal;

twenty-two, the combination pad 2 is provided with an anti-alpha-HCG antibody, the detection line T132 is the nitrocellulose membrane 3 coated with an H-HCG specific antibody B152, the detection line T233 is the nitrocellulose membrane 3 coated with an anti-beta-HCG antibody, and the quality control C line 34 is a goat antibody which is coated on the nitrocellulose membrane 3 and is anti-mouse polyclonal;

twenty three, the combination pad 2 is provided with an anti-beta-HCG antibody, the detection line T132 is formed by coating an H-HCG specific antibody B207 on the nitrocellulose membrane 3, the detection line T233 is formed by coating an H-HCG specific antibody B152 on the nitrocellulose membrane 3, and the quality control C line 34 is a goat antibody which is coated on the nitrocellulose membrane 3 and resists mouse polyclonal;

twenty-four, the combination pad 2 has anti-beta-HCG antibody, the detection line T132 is the nitrocellulose membrane 3 coated with H-HCG specific antibody B152, the detection line T233 is the nitrocellulose membrane 3 coated with H-HCG specific antibody B207, and the quality control C line 34 is the goat antibody of anti-mouse polyclonal coated on the nitrocellulose membrane 3.

Twenty five, the combination pad 2 is provided with an anti-beta-HCG antibody, the detection line T132 is formed by coating an anti-alpha-HCG antibody on the nitrocellulose membrane 3, the detection line T233 is formed by coating an H-HCG specific antibody B207 on the nitrocellulose membrane 3, and the quality control C line 34 is formed by coating a goat antibody which is anti-mouse polyclonal and is coated on the nitrocellulose membrane 3.

Twenty six, the combination pad 2 is provided with an anti-beta-HCG antibody, the detection line T132 is formed by coating an anti-alpha-HCG antibody on the nitrocellulose membrane 3, the detection line T233 is formed by coating an H-HCG specific antibody B152 on the nitrocellulose membrane 3, and the quality control C line 34 is formed by coating a goat antibody which is anti-mouse polyclonal and is coated on the nitrocellulose membrane 3.

Twenty-seven, the combination pad 2 is provided with an anti-beta-HCG antibody, the detection line T132 is formed by coating an H-HCG specific antibody B207 on the nitrocellulose membrane 3, the detection line T233 is formed by coating an anti-alpha-HCG antibody on the nitrocellulose membrane 3, and the quality control C line 34 is formed by coating a goat antibody which is anti-mouse polyclonal and is coated on the nitrocellulose membrane 3.

Twenty-eight, the combination pad 2 has anti-beta-HCG antibody, the detection line T132 is the nitrocellulose membrane 3 coated with H-HCG specific antibody B152, the detection line T233 is the nitrocellulose membrane 3 coated with anti-alpha-HCG antibody, and the quality control C line 34 is the goat antibody coated on the nitrocellulose membrane 3 for anti-mouse polyclonal.

The test paper for rapid immunodiagnosis of H-HCG can also be made into a card or a pen. When the form of card, pen is made to the binary channels mode, the test paper overcoat has a shell, design on the shell with sample pad 1 add liquid pad 21 nitrocellulose membrane 3 inhale appearance pad 4 respectively corresponding application of sample hole 7, application of liquid hole 8, nitrocellulose membrane window 9, inhale appearance pad window 10 (as shown in figure 5).

The first embodiment is as follows:

the preparation method of the H-HCG rapid immunodiagnosis chromatography test paper comprises the following steps:

the main raw materials are as follows:

reagent: chloroauric acid, trisodium citrate, antibodies against alpha-HCG or beta-HCG, H-HCG-specific antibodies B207 or B152; BSA, trehalose, NaCl, Na2HPO4 & 12H2O, NaH2PO4 & 2H2O, boric acid, borax, Tween-20, TritonX-100, sucrose and ultrapure water;

consumable material: nitrocellulose membrane (NC membrane), 8975 glass fiber, GF-06 glass fiber, water absorption pad, bottom plate, volumetric flask, conical flask, EP pipe, rifle head.

Firstly, preparation before preparation:

preparation of 1.12% citric acid trinor solution

0.105g of trinNa citrate is weighed into a 15ml EP tube, 5ml of pure water is added, and the mixture is uniformly mixed until the trinNa citrate is completely dissolved, so that 2 percent solution of trinNa citrate is obtained.

And (3) putting 40ml of pure water into a 50ml volumetric flask, adding 0.5ml of 1% chloroauric acid solution into the volumetric flask, fixing the volume to 50ml by using the pure water, and uniformly mixing to obtain the 0.01% chloroauric acid solution. Pouring 50ml of 0.01% chloroauric acid solution into a 100ml conical flask, placing the conical flask on an electric furnace, heating to boil by 3-4 steps, sucking 380 mu L of 2% citric acid trinNa solution by using a pipette, quickly pouring the solution into the boiling chloroauric acid solution, and continuously heating to finish the preparation of 40-55nm colloidal gold.

1.2 PBS buffer preparation:

weighing Na₂HPO₄·12H₂O 1.615-2g，NaH₂PO₄·2H₂0.225-5g of O and 4.0-5.0g of NaCl are put into a beaker, 400ml of ultrapure water is added for dissolving to reach the constant volume of 500ml, and the mixture is filtered by a 0.22 mu m filter membrane for standby.

1.3 preparing a re-solution:

weighing 1g of BSA and 10-13g of sucrose in a beaker, adding 70ml of PBS for dissolving, adding 0.08-0.1ml of TritonX-100, uniformly mixing, fixing the volume to 100ml, and filtering with a 0.22 mu m filter membrane for later use.

2. Determination of optimum pH:

1mL of colloidal gold was put in 6 1.5mL LEP tubes, and 0.2M K was added to each tube₂CO₃1 μ L, 2 μ L, 3 μ L, 5 μ L, 7 μ L, 10 μ L corresponding to pH of about 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, respectively, adding 150-₂CO₃The color of the colloidal gold in the EP tube with the addition amount of 1-3 μ L is still purple red, the color of other tubes is blue, and the optimum pH is 7.0-8.0.

3. Determination of minimum antibody labeling amount:

taking 4 EP tubes, respectively adding 1mL of colloidal gold into each tube, and respectively adding K into each tube₂CO₃2-4 mu L of the mixture is mixed evenly, 30 mu L (3 mu g), 50 mu L (5 mu g), 70 mu L (7 mu g) and 100 mu L (10 mu g) of 0.08-0.1mg/ml antibody solution are added into the mixture, the mixture is mixed evenly, the mixture is kept still at 37 ℃ for 20min, 10 percent NaCl solution is added into each tube, the mixture is mixed evenly and kept still for observing the color change, the color of the colloidal gold in an EP tube with the antibody adding amount of 7-10 mu g is not changed into purple red obviously, the color of the colloidal gold in an EP tube with the antibody adding amount of 3 and 5 mu g is changed into blue, and the minimum antibody adding amount of 1ml of the colloidal gold is indicated to be 5-8 mu g.

4. Preparing a gold-labeled antibody:

respectively putting 1mL of 40-55nm colloidal gold into an EP tube, and respectively adding K into the tube₂CO₃And 2-4 mu L of the solution is mixed evenly, 70 mu L of 0.08-0.1mg/ml antibody is added into an EP tube and mixed evenly, the mixture is placed at 37 ℃ for 20min, 100 mu L of the mixture is taken out from the tube and placed into a 500 mu LEP tube, 10 mu L of 10% NaCl solution is added into the mixture and mixed evenly, the mixture is kept still and is observed to be red, and the subsequent test can be carried out. Adding 100 μ L of 5% PEG solution into 40-55nm colloidal gold EP tube, standing at 37 deg.C for 2 hr for sealing, centrifuging at 4 deg.C 10000g for 20min, discarding supernatant, redissolving with 100 μ L of redissolution, and storing at 4 deg.C for use.

5、

Preparing a 0.2M boric acid solution: weighing 12.37g of boric acid in a beaker, adding 800ml of ultrapure water for dissolving, then fixing the volume to 1L, and filtering with a 0.22 mu m filter membrane for later use.

Preparing 0.05M borax solution: 19.07g of borax is weighed into a beaker, 800ml of ultrapure water is added for dissolving, the volume is determined to be 1L, and the solution is filtered by a filter membrane with the diameter of 0.22 mu m for later use.

Preparing a 10mM pH8.0 boric acid-borax buffer solution: respectively taking 3ml of 0.05M borax solution and 7ml of 0.2M boric acid solution, adding 190ml of ultrapure water into a beaker, and uniformly mixing for later use.

Preparing a 10mM pH8.4 boric acid-borax buffer solution: respectively taking 4.5ml of 0.05M borax solution and 5.5ml of 0.2M boric acid solution, adding 190ml of ultrapure water, and uniformly mixing for later use.

Second, preparation method

Preparation of sample pad 1:

preparing a sample solution, weighing 0.5g +/-0.05 g BSA in a 100ml beaker, adding 70ml 10mM pH8.4 borate buffer solution for dissolution, adding 0.3ml Tween-20, uniformly mixing, diluting to 100ml, and filtering with a 0.22 mu m filter membrane for later use;

then placing the glass fiber membrane cut into strips with the width of 0.7cm and 3.2cm in a sample solution, soaking for 4 hours at room temperature, drying at 37 ℃, and storing in a drying dish for later use;

② preparing the bonding pad 2:

preparing a treatment solution for the bonding pad 2: weighing 0.1g +/-0.05 g BSA and 0.5g +/-0.05 g trehalose in a 100ml beaker, adding 70ml 10mM pH8.0 borate buffer solution for dissolving, adding 0.3ml Tween-20, uniformly mixing, fixing the volume to 100ml, and filtering with a 0.22 mu m filter membrane for later use;

preparation of gold-labeled antibody: adding 1mL of 40-55nm colloidal gold into an EP tube, and adding 2 μ L of 0.2M K₂CO₃Mixing the solution, adding 70 μ L of 0.08-0.1mg/ml antibody solution, mixing, standing at 37 deg.C for 20min, adding 100 μ L of 10% BSA solution (final concentration of 1%), mixing, standing at 37 deg.C for 1.5h, centrifuging at 4 deg.C and 12000g for 20min, discarding supernatant, adding 1ml PBS for redissolution, centrifuging at 4 deg.C and 12000g for 20min, discarding supernatant, adding 100 μ L redissolution for redissolution, and storing at 4 deg.C;

cutting the raw material of the bonding pad 2 into strips with the width of 0.7cm, placing the strips in the treatment solution of the bonding pad 2, soaking for 4 hours at room temperature, drying at 37 ℃, spraying a gold-labeled antibody on the soaked raw material of the bonding pad 2 by using a film-scratching and gold-spraying machine according to the amount of 8-15 mu L/cm, drying at 37 ℃, and storing in a drying dish for later use;

③ coating the nitrocellulose membrane by 3: preparing a coating buffer solution: weighing 3g of trehalose in a beaker, adding 80-100ml of PBS for dissolving, adding 3-5ml of methanol, uniformly mixing, fixing the volume to 100ml, and filtering with a 0.22 mu m filter membrane for later use;

coating detection line T132: the antibody is diluted to 0.5-2mg/ml by using a coating buffer solution, drawn on the nitrocellulose membrane 3 according to the amount of 0.9-1.5 mu L/cm, dried at 37 ℃ and stored in a drying dish for later use.

Coating detection line T233: diluting to 0.5-2mg/ml with coating buffer antibody, scratching on the nitrocellulose membrane 3 according to the amount of 0.9-1.3 μ L/cm, drying at 37 ℃, and storing in a drying dish for later use.

Coated quality control C-line 34: diluting the goat anti-mouse polyclonal antibody to 1.0-1.5mg/ml by using a coating buffer solution, scratching the diluted goat anti-mouse polyclonal antibody on the nitrocellulose membrane 3 according to the amount of 0.7-1.4 mu L/cm, drying at 37 ℃ and storing in a drying dish for later use;

fourthly, preparing the test paper:

preparing single-channel test paper:

in a drying chamber with the temperature of 20-30 ℃ and the humidity of less than 30-40%, taking the support plate 5, and placing the coated nitrocellulose membrane 3 in the middle of the support plate 5 for adhesion;

overlapping and adhering a combination pad 2 (one third of the combination pad 2) on one side of a detection T line 31 of the nitrocellulose membrane 3, overlapping and adhering a sample pad 1 (one tenth of the sample pad 1) on the other side of the combination pad 2, and overlapping and adhering a sample absorption pad 4 (one tenth of the sample absorption pad 4) on one side of a quality control C line 34 on the nitrocellulose membrane 3; and sticking a layer of mark film on the uppermost surface, namely successfully preparing the H-HCG rapid immunodiagnosis test paper, and finally cutting the prepared test paper into test paper strips with the width of 3-4mm by using a cutting machine. The cut test paper strip can be used singly or can be arranged in a plastic card or a bar (pen) to form the HCG-H rapid diagnosis test paper card or bar (pen);

preparing a double-channel test paper:

in a drying chamber with the temperature of 20-30 ℃ and the humidity of less than 30-40%, taking a support plate 5, and placing the coated nitrocellulose membrane 3 in the middle of the support plate 5 for adhesion;

the sample absorbing pad 4 is adhered to one side of the nitrocellulose membrane 3, one side of the sample absorbing pad 4 is in lap joint with the nitrocellulose membrane 3, the other side edge of the sample absorbing pad 4 is flush with the support plate 5, and the other side of the nitrocellulose membrane 3 is in lap joint with the sample pad 1, so that a sample to be detected sequentially passes through the sample pad 1, the nitrocellulose membrane 3 and the sample absorbing pad 4 to form a first horizontal flow channel;

respectively lapping two ends of the fiber isolation film 6 above the sample pad 1 and the nitrocellulose membrane 3, respectively lapping two ends of the combination pad 2 above the fiber isolation film 6 and the nitrocellulose membrane 3, respectively lapping two ends of the liquid adding pad 21 above the fiber isolation film 6 and the combination pad 2, respectively, so that a buffer solution sequentially passes through the liquid adding pad 21, the combination pad 2, the nitrocellulose membrane 3 and the sample absorbing pad 4 to form a second horizontal flow channel in the detection process, namely the H-HCG rapid immune diagnosis test paper is successfully prepared, finally, the prepared test paper is cut into test paper strips with the width of 4mm by a cutting machine, and the cut test paper strips can be loaded into a plastic card or a bar (pen) to form a HCG-H rapid diagnosis test paper card or bar (pen);

the two-channel concrete research process comprises the following steps: when the sample in the sample passes through the first horizontal flow channel to reach the nitrocellulose membrane 3 and then is bound with the specific antibody on the detection T-line 31, and after 10 seconds to 5 minutes, when the sample chromatography is performed on the sample suction pad 4, the buffer solution is applied to the liquid application pad 21, and the label (conjugate) in the binding pad 2 passes through the second horizontal flow channel to reach the nitrocellulose membrane 3 and is bound with the complex of the detector-specific antibody on the detection T-line 31 and then shows a color (positive/negative) visible to the naked eye. In order to facilitate or more accurately observe whether the sample sucking pad 4 has sample chromatography or not, a coloring agent or a coloring strip can be added on the sample sucking pad 4, when the sample sucking pad 4 has sample chromatography and develops color, a buffer solution can be added at the liquid adding pad 21, and the method is superior to and/or preferentially adopts a mode of adding the buffer solution at the liquid adding pad 21 after 10 seconds to 5 minutes.

The lap joint length is 2 mm; in the double-channel test strip, the buffer solution applied to the liquid adding pad 21 is different target buffer solutions selected according to different detection objects, and is used for quickly diagnosing results. Third, test paper test and result judgment

1. And (3) test strip testing:

single channel: placing the test strip into a solution to be tested (so that the sample adding hole 7 is immersed under the liquid level) for 5s, taking out and placing on a table top, reading the result after 15min, and enabling the test strip to be effective within 30 min;

double-channel: and (3) placing the test strip into a liquid to be tested (so that the sample adding hole 7 is immersed under the liquid level) for 5s, taking out and placing the test strip on a table top, dropping 50 mu LPBS into the liquid adding hole 8 after the color in the sample sucking pad window 10 is changed from white to red, reading the result after 5min, and enabling the test strip to be effective within 30 min.

2. And (5) judging a result:

as shown in fig. 1 and 3: when two red strips appear, one is positioned on the detection line T, and the other is positioned on the quality control line C34, the pregnancy is shown, or the placenta function of the pregnant woman is shown to be good, and the abortion probability is very small; when a red band appears, it is only located on the quality control C line 34, indicating no pregnancy or indicating poor placental function in pregnant women; at 5-10 minutes, no red bands appear on the quality control C-line 34, indicating an improper procedure or a test strip that has deteriorated and/or damaged, requiring a new test strip to be properly handled and/or retested.

Referring to fig. 2 and 4, when three red strips appear, one is located at the detection line T132, the other is located at the detection line T233, and the other is located at the quality control line C34, the pregnancy is shown, and the placenta of the pregnant woman has good function and the chance of abortion is very small; when two red strips appear, one is positioned on a detection line T132 or T2, and the other is positioned on a quality control line C34, the pregnancy is shown, but the placenta of the pregnant woman has poor function and has high abortion occurrence probability, and further examination and/or treatment are needed; when a red band appears, it is only located at the quality control C line 34, indicating no pregnancy; at 5-10 minutes, no red bands appear on the quality control C-line 34, indicating an improper procedure or a test strip that has deteriorated and/or damaged, requiring a new test strip to be properly handled and/or retested.

Example two

Sensitivity detection of H-HCG rapid immunodiagnosis chromatography test paper

1. 12.5mIU/ml, 25mIU/ml and 50mIU/ml HCG solution preparation: the HCG standard was reconstituted with PBS containing 0.5% BSA and formulated into 12.5mIU/ml, 25mIU/ml, 50mIU/ml HCG solutions.

2. Preparation of 110mIU/ml H-HCG solution: the H-HCG standard was reconstituted with 0.5% BSA in PBS and made up to 110 mIU/ml.

3. Single channel: placing the test strips of the same batch number into 12.5mIU/ml, 25mIU/ml, 50mIU/ml HCG solution and 110mIU/ml H-HCG solution respectively, staying for about 5s, taking out, placing on a table top, waiting for about 15min, observing the result, and detecting the result to be effective within 30 min. Repeating each detection concentration for 3 times, wherein the detection result of each solution for 3 times is positive, and the result of each solution for the second concentration can be judged to be positive;

double-channel: placing test strips of the same batch number into 12.5mIU/ml, 25mIU/ml, 50mIU/ml HCG solution and 110mIU/ml H-HCG solution respectively, staying for about 5s (enabling the sample adding hole 7 to be immersed under the liquid level), taking out and horizontally placing on a table board, adding 50 mu L PBS solution into the upper layer liquid adding hole 8 after the color in the sample sucking pad window 10 is changed from white to red, waiting for about 10min to read the result, enabling the detection result to be effective within 30min, repeating 3 times for each detection concentration, and judging that the result of the secondary concentration is positive when the detection result of each solution is positive for 3 times.

TABLE 1 Single/Dual channel sensitivity test

Note: "-" indicates invisible, "+ + + + +" indicates clearly visible, 110 ═ 110mIU/ml H-HCG solution.

From table 1, it can be seen that: when single-channel and double-channel sensitivity tests are carried out on 12.5mIU/ml, 25mIU/ml and 50mIU/ml HCG standard substances, HCG lines are normal and meet the requirements of national standards, and H-HCG lines are not led out, which indicates that the HCG standard substances and H-HCG antibodies do not have cross reaction; when the H-HCG standard substance with 110mIU/ml is tested, HCG and H-HCG lines are both out, which shows that the H-HCG standard substance has better cross reaction with anti-alpha-HCG antibody, anti-beta-HCG antibody and H-HCG antibody B207/B152.

EXAMPLE III

Specificity detection of H-HCG rapid immunodiagnosis chromatography test paper

1. Solution preparation: HCG solution 0 mIU/ml: 0.5g BSA was weighed into a beaker, and 70ml PBS buffer was added to dissolve to 100ml, and the solution was filtered through a 0.22 μm filter for further use.

2. 500mIU/ml human luteinizing hormone (hLH) solution: re-dissolving the hLH standard substance by using PBS (phosphate buffer solution) containing 0.5% BSA (bovine serum albumin), and preparing the hLH standard substance into 500mIU/ml HCG (human serum albumin) solution, namely hLH A solution; preparing the solution into 500mIU/ml HCG solution by using 25mIU/ml HCG solution, namely hLH B solution; the solution is prepared into hLH C solution with the concentration of 500mIU/ml by using 110mIU/ml H-HCG solution.

3. 1000mIU/ml human follicle stimulating hormone (hFSH) solution: re-dissolving the hFSH standard substance by using PBS solution containing 0.5 percent BSA, and preparing the hFSH standard substance into HCG solution with the concentration of 1000mIU/ml by using HCG solution with the concentration of 0mIU/ml, namely hFSH A solution; preparing the solution into a HCG solution with the concentration of 1000mIU/ml by using a HCG solution with the concentration of 25mIU/ml, namely the hFSH B solution; the solution is prepared into hFSH C solution with the concentration of 1000mIU/ml by using 110mIU/ml H-HCG solution.

4. 1000 μ IU/ml human thyrotropin (hTSH) solution: re-dissolving the hLH standard substance by using PBS solution containing 0.5 percent BSA, and preparing the hLH standard substance into HCG solution with the concentration of 1000 mu IU/ml by using HCG solution with the concentration of 0 mu IU/ml, namely hTSH A solution; preparing the hTSH solution into HCG solution with the concentration of 1000 mu IU/ml by using 25mIU/ml HCG solution, namely hTSH B solution; the hTSH C solution is prepared by using 110mIU/ml H-HCG solution, and the concentration of the hTSH C solution is 1000 mu IU/ml.

5. Negative specificity:

single channel: respectively putting test strips of the same batch number into an hLH A solution with the concentration of 500mIU/ml, an hFSH A solution with the concentration of 1000mIU/ml and an hTSH A solution with the concentration of 1000 mu IU/ml for staying for about 5s, taking out and flatly placing on a table top for waiting for about 15min, observing results, wherein the detection results are effective within 30min, repeating each concentration for 3 times, and judging that the specificity of each solution is negative if the 3 detection results of each solution are negative;

double-channel: putting the test strips of the same batch number into an hLH A solution with the concentration of 500mIU/ml, an hFSH A solution with the concentration of 1000mIU/ml and an hTSH A solution with the concentration of 1000 mu IU/ml respectively for about 5s (the sample adding hole 7 is immersed under the liquid level), taking out and horizontally placing the test strips on a table top, adding 50 mu L of PBS solution into the upper liquid adding hole 8 when the color in the window 10 of the sample sucking pad changes from white to red, waiting for about 10min to read the result, enabling the detection result to be effective within 30min, repeating the concentration 3 times respectively, and judging that the specificity of the solution is negative if the detection result of each solution for 3 times is negative.

TABLE 2 Single/Dual channel negative specificity test results

Note: "-" indicates invisible, "+ + + + +" indicates clearly visible

From table 2, it can be seen that: the single-channel and double-channel test strip has negative specificity test HCG line and H-HCG line, and both T lines have no false positive, and the index meets the national standard requirement.

6. Positive specificity:

single channel: putting the test strips of the same batch number into the Hlh B liquid and the C liquid with the concentration of 500mIU/ml, the hFSH B liquid and the C liquid with the concentration of 1000mIU/ml, the hTSSH B liquid and the C liquid with the concentration of 1000 mu IU/ml for about 5s, taking out, putting the test strips on a table board for waiting for about 15min, observing the result, and detecting the result to be effective within 30 min. Repeating the concentration for 3 times, and judging the specificity of the solution to be negative if the 3 detection results of the solutions are negative;

double-channel: putting the test strips of the same batch number into the solutions of 500mIU/ml hLH B liquid and C liquid, 1000mIU/ml hFSH B liquid and C liquid, 1000 mu IU/ml hTSSH B liquid and C liquid respectively, staying for about 5s (making the sample adding hole 7 immersed under the liquid level), taking out and horizontally placing on a table top, adding 50 mu L PBS solution into the upper layer liquid adding hole 8 after the color in the window 10 of the sample sucking pad is changed from white to red, waiting for about 10min to read the result, enabling the detection result within 30min, repeating the concentration for 3 times, and judging that the specificity of the solution is positive if the detection result of each solution for 3 times is positive.

TABLE 3 Single/Dual channel Positive specificity test results

Note: "-" indicates invisible, "+ + +" indicates visible, "+ + + +" indicates clearly visible

From table 3, it can be seen that: the HCG line and the H-HCG line of the single-channel test strip and the double-channel test strip are both positive in positive specificity test, no false negative appears in both the two T lines, and the index also meets the national standard requirement.

Example four:

stability detection of H-HCG rapid immunodiagnosis chromatography test paper

And (3) placing the test strips of the same batch number at 37 ℃ for 7d, 15d and 21d, and then testing the sensitivity, the specificity and the repeatability of the test strips respectively, wherein the sensitivity and the specificity testing method is the same as that of the second embodiment and the third embodiment.

The repeatability test method is as follows:

1. single channel: and (3) taking 20 test strips at each time point, putting the test strips into an HCG standard substance with the concentration of 25mIU/ml or an H-HCG solution with the concentration of 110mIU/ml, staying for about 5s, taking out, putting the test strips on a table board, waiting for about 15min, observing the result, and enabling the test result to be effective within 30 min.

2. Double-channel: and (3) taking 20 test strips at each time point, putting the test strips into a HCG standard substance with the concentration of 25mIU/ml or an H-HCG solution with the concentration of 110mIU/ml for about 5s (the sampling hole 7 is immersed under the liquid level), taking out and horizontally placing the test strips on a table, adding 50 mu L of PBS solution into the upper liquid adding hole 8 after the color in the window 10 of the sample sucking pad is changed from white to red, waiting for about 10min to read the result, and enabling the detection result to be effective within 30 min.

Table 437 deg.C, test results of single-channel and double-channel minimum detection limit of test strip after 21 days of destruction

Table 537 ℃ test results of test strip negative specificity after 21 days of destruction

Note: "-" indicates invisible, "+ + + + +" indicates clearly visible

TABLE 637 deg.C, test result of test strip positive specificity after 21 days of destruction

Table test paper strip repeatability test results after 737 deg.c destruction

From tables 4-7, it can be seen that: after the single-channel test strip and the double-channel test strip are damaged at 37 ℃, the sensitivity, the specificity and the repeatability are tested, the results have no false positive or false negative, the national standard requirements are met, and the test strip stability is good.

Example five:

clinical experiment of H-HCG quick immunodiagnosis chromatography test paper

The test strip is used for detecting collected clinical urine and blood according to a using method, hospital clinical examination results are compared, the hospital blood examination results show that 5 samples in 50 samples have blood HCG values smaller than 1.2mIU/ml, are diagnosed as not pregnant and negative samples, other 45 samples have blood HCG values larger than 5mIU/ml, are diagnosed as pregnant and positive samples, and 5 fetal presenting samples are diagnosed in 45 positive samples through other clinical auxiliary examinations; the urine and blood of the 50 samples are respectively detected by the test strip, 5 negative samples detected by the test strip are consistent with clinical results of a hospital and have no false positive, other 45 samples detected by the test strip are all positive and have no false negative, the HCG line coincidence rate is 100%, in addition, 5 fetal presenting termination samples detected by the test strip in the 45 positive samples are also consistent with clinical diagnosis results of the hospital, the H-HCG line coincidence rate of the fetal presenting termination detection is also 100%, and the test strip can simultaneously detect the primary diagnosis of abnormal pregnancy or abortion of normal pregnancy and fetal presenting termination. The clinical test of the test strip is shown in table 8:

TABLE 8 test strip clinical test results

Note: "-" indicates invisible, "+ + + + +" indicates clearly visible, + + indicates visible, + weakly visible

As used herein, H-HCG represents hyperglycosylated human chorionic gonadotropin.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A H-HCG rapid immunodiagnosis chromatography test paper is characterized in that: including sample pad (1), the parcel that connects gradually have nitrocellulose membrane (3) of detecting T line (31) and matter accuse C line (34), inhale kind pad (4), just sample pad (1) nitrocellulose membrane (3) inhale kind pad (4) overlap joint in proper order, paste on a backup pad (5).

2. The H-HCG rapid immunodiagnostic chromatographic strip of claim 1, wherein:

the test strip also comprises a combination pad (2), and the combination pad (2) is arranged between the sample pad (1) and the nitrocellulose membrane (3), so that the sample pad (1), the combination pad (2), the nitrocellulose membrane (3) and the sample sucking pad (4) form a single-channel test strip.

3. The H-HCG rapid immunodiagnostic chromatographic strip of claim 1, wherein:

still include a fibre barrier film (6), a combination pad (2) and one add liquid pad (21), just fibre barrier film (6) both ends overlap joint respectively sample pad (1) nitrocellulose membrane (3) top, combination pad (2) both ends overlap joint respectively fibre barrier film (6) nitrocellulose membrane (3) top, add liquid pad (21) both ends overlap joint respectively fibre barrier film (6) combination pad (2) top makes sample pad (1) with fibre barrier film (6), combination pad (2) with add liquid pad (21) nitrocellulose membrane (3) inhale appearance pad (4) and constitute the binary channels test paper.

4. The H-HCG rapid immunodiagnostic chromatographic strip of claim 2 or 3, wherein: an H-HCG specific antibody B207 or B152 is arranged on the binding pad (2), the H-HCG specific antibody B152 or B207 is coated on the nitrocellulose membrane (3) T line, and the quality control C line (34) contains a goat anti-mouse polyclonal antibody;

or,

an anti-alpha-HCG or anti-beta-HCG antibody, or an anti-complete HCG antibody, or an H-HCG specific antibody B207 or B152 is arranged on the binding pad (2); the detection T line (31) consists of a detection line T1(32) and a detection line T2(33), wherein the detection line T1(32) is coated with an anti-alpha-HCG or beta-HCG antibody, or an anti-complete HCG antibody, the detection line T2(33) is coated with an H-HCG specific antibody B152 or B207, or the detection line T1(32) is coated with an H-HCG specific antibody B152 or B207, the detection line T2(33) is coated with an anti-alpha-HCG or beta-HCG antibody, or an anti-complete HCG antibody,

or,

suitable for an immune competition method, the nitrocellulose membrane (3) is coated with H-HCG antigen on a T line or vice versa, the binding pad (2) is provided with the H-HCG antigen, and the T line is coated with H-HCG specific antibody.

5. A preparation method of H-HCG rapid immunodiagnosis chromatography test paper is characterized by comprising the following steps: the method comprises the following steps:

preparation of a sample pad (1): preparing a sample pad treatment solution, and then placing the cut glass fiber film or non-woven fabric in the sample pad treatment solution for soaking at room temperature and drying for later use;

preparing the bonding pad (2): preparing a bonding pad treatment solution and a gold-labeled antibody, placing the bonding pad raw material in the bonding pad treatment solution for soaking at room temperature and drying, spraying the gold-labeled antibody on the soaked bonding pad raw material by using a film-scratching gold-spraying machine, and drying for later use;

③ coating the nitrocellulose membrane (3): preparing a coating buffer solution, and respectively diluting a goat anti-mouse polyclonal antibody, an anti-alpha-HCG or beta-HCG antibody, or an anti-complete HCG antibody, an H-HCG specific antibody B207 or B152 to 0.1-20mg/ml with the coating buffer solution to obtain a quality control C line (34), a detection line T1(32), a detection line T2(33) or a quality control C line (34), a detection line T2(33), a detection line T1(32), or a quality control C line (34) and a detection T line (31);

fourthly, preparing the test paper:

single-channel test paper: in a drying chamber at normal temperature, taking the supporting plate (5), firstly attaching the coated nitrocellulose membrane (3) to the middle part of the supporting plate (5), then respectively attaching the combination pad (2) and the sample sucking pad (4) to two sides of the nitrocellulose membrane (3), attaching the sample pad (1) to one side of the combination pad (2) opposite to the nitrocellulose membrane (3), and finally cutting the supporting plate (5) by using a cutting machine;

double-channel test paper: the sample sucking pad (4) is adhered to one side of the nitrocellulose membrane (3), one side of the sample sucking pad (4) is overlapped with the nitrocellulose membrane (3), the edge of the other side of the sample sucking pad is flush with the supporting plate (5), and the other side of the nitrocellulose membrane (3) is overlapped with the sample pad (1), so that a sample to be detected sequentially passes through the sample pad (1), the nitrocellulose membrane (3) and the sample sucking pad (4) to form a first horizontal flow channel;

will fibre barrier film (6) both ends overlap joint respectively sample pad (1) nitrocellulose membrane (3) top, combination pad (2) both ends overlap joint respectively fibre barrier film (6) nitrocellulose membrane (3) top, add liquid pad (21) both ends overlap joint respectively fibre barrier film (6) combination pad (2) top for buffer solution loops through in the testing process add liquid pad (21) combination pad (2) nitrocellulose membrane (3) inhale appearance pad (4) and form second horizontal flow channel, adopt the cutter to cut at last backup pad (5).

6. The method of claim 5, wherein: in the step (i), the preparation method of the sample treatment solution comprises the following steps: weighing 0.5g +/-0.05 g BSA in a beaker, adding 70ml 10mM borate buffer solution with pH8.4 for dissolving, adding Tween-20, mixing uniformly, fixing the volume, and filtering with a filter membrane for later use.

7. The method of claim 5, wherein: in the second step, the preparation method of the combined pad treatment solution comprises the following steps: weighing 0.1g +/-0.05 g BSA and 0.5g +/-0.05 g trehalose in a beaker, adding 70ml 10mM borate buffer solution with pH8.0 for dissolving, adding Tween-20, uniformly mixing, and filtering with a filter membrane for later use; the gold-labeled antibody contains an anti-alpha-HCG or anti-beta-HCG antibody, or an anti-complete HCG antibody, or an H-HCG specific antibody B207 or B152.

8. The method of claim 5, wherein: in the third step, the preparation method of the coating buffer solution comprises the following steps: dissolving trehalose in PBS, adding methanol, mixing, diluting to desired volume, and filtering with filter membrane.

9. The production method according to claims 6 to 8, characterized in that: the sample pad (1) is made of glass fiber film or non-woven fabric; the sample absorbing pad (4) is made of water absorbing filter paper.

10. The production method according to claims 6 to 8, characterized in that: the binding pad (2) is composed of 5-100nm colloidal gold or 5-800nm nano-materials or latex particles or carbon particles or other luminescent dyes or enzyme-bound anti-HCG antibodies or H-HCG specific antibodies B207 or B152, or quoted or bound with avidin and biotin; the binding pad (2) is added with avidin or biotin in colloidal gold or cellulose nanoparticles labeled with specific antibodies, or colloidal gold-cellulose nanoparticles labeled with specific antibodies, or both.