CN106248975A - A kind of H HCG Rapid immunodiagnosis chromatographic test paper and preparation method thereof - Google Patents
A kind of H HCG Rapid immunodiagnosis chromatographic test paper and preparation method thereof Download PDFInfo
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- CN106248975A CN106248975A CN201610541351.2A CN201610541351A CN106248975A CN 106248975 A CN106248975 A CN 106248975A CN 201610541351 A CN201610541351 A CN 201610541351A CN 106248975 A CN106248975 A CN 106248975A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical 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
- G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
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
Technical field
The present invention relates to technical field of medical detection, specifically a kind of H-HCG Rapid immunodiagnosis chromatographic test paper and
Its preparation method.
Background technology
Human chorionic gonadotropin (human chorionic gonadotrophin is called for short HCG), is to be nourished by Placenta Hominis
Confluent monolayer cells is secreted, molecular weight is the glycoprotein hormones of 36700, and it is made up of α and β bis-subunit.HCG is clinical pregnancy and pregnant
The label that relevant disease of being pregnent is the most frequently used in checking, its biological immunology feature is mainly determined by β subunit, for avoiding anti-HCG to resist
There is cross reaction with other polypeptide hormones such as FSH, LH and TSH etc. in body, although complete HCG is found to be during gestation
Common form, but HCG exists in blood, urine in different forms, and other hypotype of HCG or fragment are also presented in anemia of pregnant woman's
In blood, urine and saliva, these hypotypes or fragment include H-hCG (H-HCG), free
HCG-α unit, free HCG-β unit, HCG-β core fragment and breach HCG, they are respectively provided with different biological functions.
But, the detection instrument of now widely used detection people HCG is HCG colloidal gold immunochromatographydetection detection test paper, and uses anti-
α and the β chain of HCG or the method for β HCG monoclonal antibody detection pregnancy serum total HCG concentration, measured HCG value is respectively less than
The actual summation of various HCG components, and can not completely and all types or hypotype or fragment HCG combine, can not provide
The information of various hypotypes, particularly their biological function and relevant clinical meaning.
Early pregnancy can be divided into normal pregnancy and abnormal gestation, and abnormal gestation includes ectopic pregnancy and spontaneous abortion,
Sickness rate is respectively 1% and about 10-15% in China, and in ascendant trend year by year, is the major causes of death of pregnant and lying-in women
One of, have become as the health problem that Women of childbearing age is important.In order to determine ectopic pregnancy and spontaneous abortion, although we are to anemia of pregnant woman
Carry out progesterone and the detection by quantitative of HCG level, but the most also do not have a kind of screening technique the most immediately to examine
Disconnected.Also care diagnostic method distinguishes the most pregnant and abnormal gestation in early days the most quickly and accurately.On Present clinical
General method is, one is ultrasonic examination, and two is the existence that HCG multiplication test determines ectopic pregnancy.By Transvaginal Ultrasound,
See and whether there is embryo's capsule to judge that embryo exists, be then to see the gestation existence with or without dystopy by abdominal ultrasonic.But use
Ultrasonic check that embryo, the method for embryo's capsule are suitable for 5-8 week gestation (about after menolipsis 1-4 week), when there being ectopic pregnancy disease in early days
Often have already passed by ultrasonographic opportunity when the anemia of pregnant woman of shape goes to a doctor, another relied on HCG multiplication test and analyze, as pregnant in two days
In woman's blood, HCG level does not double, then show ectopic pregnancy.Contrary spontaneous abortion person cannot use above-mentioned two kinds of analysis methods
?.The multiplication factor measuring every two days HCG has become the department of obstetrics and gynecology inspection method of standard, in two days decline HCG level and
Do not double and usually show Pregnancy failure.But the method has its weak point, one anemia of pregnant woman to need medical secondary, its two needs are taken out
More than the horizontal secondary of blood secondary detection HCG, its three testing result is restricted, and the sensitivity of the most different document report detections is
62-78%, false positive rate is up to 26-40%.
Detection H-hCG (H-HCG) level, it is possible to well monitoring or differentiation are the most pregnant
Be pregnent for normal pregnancy and abnormal gestation because the H-HCG normocrinic a kind of hormone that is First Trimester, its function is for completing
Placenta Hominis is to the implantation of endometrium, and therefore for normal pregnancy, the deficiency of H-HCG secretion can make Placenta acrreta and growth barrier
Hinder, cause placenta development bad and cause miscarriage.Research display, (according to last menstrual period) serum H-HCG prediction when pregnant 4～7 weeks
The efficiency of miscarriage checks higher than conventional H CG.The ratio of the H-HCG and total HCG at (according to LH peak) during the detection gestation such as Sasaki about 7 days
Value predicts miscarriage, it is thus achieved that the positive predictive value of 100% and the negative predictive value of 75%.Another is to external fertilization (IVF)
The research of gravid woman shows, after Embryonic limb bud cell H-HCG detection prediction IVF after 3 weeks, pregnancy outcome's effect is better than the inspection of conventional H CG
Look into, and to distinguish miscarriage reason be Embryonic limb bud cell failure or clinical spontaneous abortion, the serum of postoperative to IVF the 6th day such as Strom
Detection with uterine cancer cell is it was also found that H-HCG has sensitivity and the specificity of 100% to the diagnosis of successful pregnancy.And can differentiate
Biochemical pregnancies.Cole finds in nearest research, and the Serum HCG concentration of successful pregnancy and spontaneous abortion person does not has the biggest difference
Different, but spontaneous abortion gravid woman's serum H-HCG concentration is significantly lower than successful pregnancy person, and the serum H-HCG water of biochemical pregnancies
Flat then lower.The detection of H-HCG has its superiority and weight at aspects such as early pregnancy, ectopic pregnancy, early abortion, tumor monitorings
The clinical meaning wanted.Therefore, study H-HCG, how to be applied in product, prepare a kind of detection speed fast and convenient
Product extremely urgent.
Summary of the invention
The present invention is to solve above-mentioned technological deficiency, it is provided that a kind of H-HCG Rapid immunodiagnosis chromatographic test paper and preparation thereof
Method, the method is convenient, fast, accuracy is high, and this reagent paper be suitable in early days normal pregnancy and the diagnosis of abnormal gestation and
Differential Diagnosis, has important clinical meaning at aspects such as early pregnancy, ectopic pregnancy, early abortion, tumor monitorings.
One aspect of the present invention, it is provided that a kind of H-HCG Rapid immunodiagnosis reagent paper, including: the sample pad that is sequentially connected with, is coated
There are detection T line and the nitrocellulose filter of Quality Control C line, sample suction pad, and described sample pad, described nitrocellulose filter, described suction
Sample pad overlaps successively, is pasted onto in a gripper shoe；
Preferably, H-HCG Rapid immunodiagnosis reagent paper also includes a pad, and described pad is arranged on described sample
Between pad and described nitrocellulose filter so that described sample pad and described pad, described nitrocellulose filter, described suction sample
Pad constitutes single channel reagent paper；
Or
H-HCG Rapid immunodiagnosis reagent paper also includes that a fiber isolated film, a pad and add fluid cushion, and described fibre
Dimension isolated film two ends are overlapped on above described sample pad, described nitrocellulose filter respectively, and described pad two ends are taken respectively
Be connected on above described fiber isolated film, described nitrocellulose filter, described in add fluid cushion two ends be overlapped on respectively described fiber every
Above scrapping off film, described pad so that described sample pad and described fiber isolated film, described pad, described in add fluid cushion,
Described nitrocellulose filter, described sample suction pad constitute dual pathways reagent paper.
Further,
H-HCG heterogenetic antibody B207 or B152 it is provided with, the described inspection on described nitrocellulose filter on described pad
Surveying T line and be coated with H-HCG specific antibody B152 or B207, described Quality Control C line contains sheep anti mouse polyclonal antibody；
Or,
It is provided with anti alpha-HCG or the antibody of β-HCG, or anti-complete HCG antibody on described pad, or is provided with H-
HCG specific antibody B207 or B152；Detection T line by detecting line T1, detection line T2 forms, and described detection line T1 is coated with anti-
Be coated with on α-HCG or the antibody of β-HCG, or anti-complete HCG antibody, detection line T2 H-HCG specific antibody B152 or
Be coated with H-HCG specific antibody B152 or B207 on B207, or detection line T1, detection line T2 is coated with anti alpha-HCG or
The antibody of β-HCG, or anti-complete HCG antibody；It is suitable for Immune competition method, described nitrocellulose filter (3) T line is coated with
H-HCG antigen or contrary, pad (2) is provided with H-HCG antigen, T line is coated with H-HCG specific antibody.
Second aspect present invention, it is provided that the preparation side of a kind of H-HCG Rapid immunodiagnosis chromatographic test paper described in first aspect
Method, comprises the steps:
1., the preparation of sample pad: prepare sample pad treatment fluid, then the glass fibre membrane after cutting out or non-woven fabrics are placed in
Soaking at room temperature in sample pad treatment fluid, drying, standby；
2., the preparation of pad: prepare pad treatment fluid, gold labeling antibody, pad raw material is placed in pad and processes
Soaking at room temperature, drying in liquid, then with draw film metal spraying machine by gold labeling antibody be sprayed on soaking after pad raw material, dry for standby；
3., nitrocellulose filter is coated: preparation is coated buffer, dilutes sheep anti mouse Anti-TNF-α respectively with being coated buffer
Body, anti alpha-HCG or the antibody of β-HCG, or anti-complete HCG antibody (or they can be monoclonal antibody, polyclonal antibody
Or the multiple fragment of antibody), H-HCG monoclonal antibody B207 or B152 to 0.1-20mg/ml, it is thus achieved that Quality Control C line, detection line
T1, detection line T2 or Quality Control C line, detection line T2, detection line T1 or Quality Control C line, detection T line；
4., the preparation of reagent paper:
Single channel reagent paper: in the hothouse of room temperature, takes described gripper shoe, first by above-mentioned coated described celluloid
Film fits in the middle part of described gripper shoe, then pastes described pad, described sample suction pad respectively in nitrocellulose filter both sides, and
Paste described sample pad in the side that described pad is relative with described nitrocellulose filter, finally use cutter to shear described
Gripper shoe；
Dual pathways reagent paper: described sample suction pad is pasted in described nitrocellulose filter side, and described sample suction pad side is with described
Nitrocellulose filter overlaps, and opposite side edge is concordant with described gripper shoe, described nitrocellulose filter opposite side and described sample
Pad overlap joint so that sample to be tested passes sequentially through described sample pad, described nitrocellulose filter, described sample suction pad form the first level
Flow channel；
Described fiber isolated film two ends are overlapped on above described sample pad, described nitrocellulose filter respectively, described
Pad two ends are overlapped on above described fiber isolated film, described nitrocellulose filter respectively, described in add fluid cushion two ends respectively
Be overlapped on above fiber isolated film, described pad so that during detection buffer pass sequentially through described in add fluid cushion, described
Pad, described nitrocellulose filter, described sample suction pad form the second bottom horizontal flow sheet passage, finally use cutter to shear described
Gripper shoe.
Preferably, described step 1. in, the preparation method of sample treatment solution is: weigh 0.5g ± 0.05gBSA in beaker
In, add 70ml 10mM pH8.4 borate buffer solution and dissolve, add tween 20, mix, standby after constant volume, membrane filtration.
Preferably, step 2. in, the preparation method of pad treatment fluid is: weigh 0.1g ± 0.05gBSA, 0.5g ±
0.05g trehalose, in beaker, adds 70ml 10mM pH8.0 borate buffer solution and dissolves, and adds tween 20, mixing, filter membrane
After filtration standby；Containing anti alpha-HCG or the antibody of β-HCG in described gold labeling antibody, or anti-complete HCG antibody, or H-HCG is different
Property antibody B207 or B152.
Described step 3. in, the preparation method being coated buffer is: take trehalose in beaker, adds after PBS dissolves again
Add methanol, standby after mixing, constant volume, membrane filtration.
Further, described sample pad material is glass fibre membrane or non-woven fabrics；Described sample suction pad is made up of absorbent filter.
Further, described pad (2) is by 5-100nm gold colloidal or 5-800nm nano material or latex particle or carbon
Granule or other luminescent dye or enzyme combine Anti-HCG antibody or H-HCG antibody composition, or quote or combine Avidin, biotin；
Described pad (2) is marked with gold colloidal or the cellulose nano-particle of specific antibody, or is marked with specific antibody
Gold colloidal cellulose nano-particle, or the two is added with Avidin or biotin.
One H-HCG Rapid immunodiagnosis chromatographic test paper of the present invention and preparation method thereof, its advantage is:
1., be coated on pad, nitrocellulose filter H-HCG heterogenetic antibody B207 or B152 or anti alpha-HCG or β-
The antibody of HCG, or the design of anti-complete HCG antibody, can be by the embodiment such as blood, urine, saliva etc. of human body H-HCG content
The detection of sample knows that the most conceived or anemia of pregnant woman the placental function of detected person is the best and predicts miscarriage etc.；And
This H-HCG reagent paper is applicable to First Trimester, and after particularly having sexual intercourse, 5-7 days or same day delayed menstruaion can start inspection for first 1-2 days
Survey, be available for medical institutions at different levels and women gestation is carried out tentative diagnosis and women's oneself's detection at home；
2., this diagnostic reagent utilizing immune chromatography test paper method quickly to detect, be particularly suitable in early days normal pregnancy and
The diagnosis and differential diagnosis of abnormal gestation, has high specificity, sensitivity height, has safety, easily preservation, good stability and answer
By the advantage such as convenient, in terms of medical science quick diagnosis, health examination, generaI investigation and home diagnostic, the medical health of women and excellent
Raw good child-rearing aspect has bigger applied value and clinical meaning.
Accompanying drawing explanation
Fig. 1, Fig. 2 are a kind of H-HCG Rapid immunodiagnosis chromatographic test paper single-pass configuration schematic diagram；
Fig. 3, Fig. 4 are a kind of H-HCG Rapid immunodiagnosis chromatographic test paper channel structure schematic diagram；
Fig. 5 is dual pathways overcoat housing structural representation；
Wherein:
1, sample pad, 2, pad, 21, add fluid cushion, 3, nitrocellulose filter, 31, detection T line, 32, detection line T1,33,
Detection line T2,34, Quality Control C line, 4, sample suction pad, 5, gripper shoe, 6, fiber isolated film, 7, well, 8, liquid filling hole, 9, nitric acid
Cellulose membrane form, 10, sample suction pad form.
Detailed description of the invention
A kind of H-HCG Rapid immunodiagnosis chromatographic test paper, including the sample pad 1 being sequentially connected with, be coated with detection T line 31 with
The nitrocellulose filter 3 of Quality Control C line 34, sample suction pad 4, and described sample pad 1, described nitrocellulose filter 3, described sample suction pad 4
Overlap successively, be pasted onto in a gripper shoe 5.
Preferably, H-HCG Rapid immunodiagnosis chromatographic test paper: also include a pad 2, and described pad 2 is arranged on
Between described sample pad 1 and described nitrocellulose filter 3 so that described sample pad 1 and described pad 2, described cellulose nitrate
Element film 3, described sample suction pad 4 constitute single channel reagent paper (as shown in Figure 1 and Figure 2).
Preferably, H-HCG Rapid immunodiagnosis chromatographic test paper: also include fiber isolated film 6, pad 2 and
Add fluid cushion 21, and described fiber isolated film 6 two ends be overlapped on above described sample pad 1, described nitrocellulose filter 3 respectively,
Described pad 2 two ends are overlapped on above described fiber isolated film 6, described nitrocellulose filter 3 respectively, described in add fluid cushion 21
Two ends are overlapped on above fiber isolated film 6, described pad 2 respectively so that described sample pad 1 and described fiber isolated film
6, described pad 2 and described add fluid cushion 21, described nitrocellulose filter 3, described sample suction pad 4 constitute dual pathways reagent paper (such as figure
3, shown in Fig. 4)；
Further,
It is provided with H-HCG heterogenetic antibody B207 or B152 on described pad 2, described nitrocellulose filter 3 is coated with
H-HCG specific antibody B152 or B207, described Quality Control C line 34 is containing sheep anti mouse polyclonal antibody；
Or,
Anti alpha-HCG or the antibody of β-HCG, or anti-complete HCG antibody it is provided with on described pad 2, or be provided with
H-HCG specific antibody B207 or B152；Detection T line 31 is made up of detection line T1 32, detection line T2 33, and described detection line
T1 32 is coated with on anti alpha-HCG or the antibody of β-HCG, or anti-complete HCG antibody, detection line T2 33 and is coated with H-HCG spy
It is coated with H-HCG specific antibody B152 or B207 on heterogenetic antibody B152 or B207, or detection line T1 32, detects line T2
Anti alpha-HCG or the antibody of β-HCG, or anti-complete HCG antibody it is coated with on 33；
Or,
It is suitable for Immune competition method, described nitrocellulose filter 3T line is coated with H-HCG antigen or contrary, pad 2
On be provided with H-HCG antigen, T line is coated with H-HCG specific antibody.
Preferably, described sample pad 1 material is glass fibre membrane or non-woven fabrics；Described sample suction pad 4 is made up of absorbent filter.
Preferably, described pad 2 is by 5-100nm gold colloidal or 5-800nm nano material or latex particle or carbon granule
Or other luminescent dye or enzyme combine Anti-HCG antibody or H-HCG antibody composition, or quote or combine Avidin, biotin；Described
Pad 2 is marked with gold colloidal or the cellulose nano-particle of specific antibody, or is marked with the colloid of specific antibody
Gold cellulose nano-particle, or the two is added with Avidin or biotin.
Described detection T line 31 has sessile antibody or H-HCG antigen or added with biotin or Avidin.
In described above, the consumption of H-HCG specific antibody B207 and B152 0.01-100mg/ml, anti alpha-HCG or β-
The antibody of HCG, or the consumption of anti-complete HCG antibody is at 0.01-100mg/ml.
One H-HCG Rapid immunodiagnosis chromatographic test paper of the present invention, specifically has a following form:
One, pad 2 is containing H HCG specific antibody B207, and detection T line 31 is for being coated with on nitrocellulose filter 3
H HCG specific antibody B152, Quality Control C line 34 is the sheep anti mouse polyclonal antibody being coated on nitrocellulose filter 3；
Two, pad 2 is containing H HCG specific antibody B207, and detection T line 31 is for being coated with on nitrocellulose filter 3
H HCG specific antibody B207, Quality Control C line 34 is the sheep anti mouse polyclonal antibody being coated on nitrocellulose filter 3；
Three, pad 2 is containing H HCG specific antibody B152, and detection T line 31 is for being coated with on nitrocellulose filter 3
H HCG specific antibody B207, Quality Control C line 34 is the sheep anti mouse polyclonal antibody being coated on nitrocellulose filter 3；
Four, pad 2 is containing H HCG specific antibody B152, and detection T line 31 is for being coated with on nitrocellulose filter 3
H HCG specific antibody B152, Quality Control C line 34 is the sheep anti mouse polyclonal antibody being coated on nitrocellulose filter 3；
Five, pad 2 is containing H HCG specific antibody B207, and detection line T1 32 is for being coated with on nitrocellulose filter 3
Anti alpha-HCG antibody, detection line T2 33 is for being coated with H HCG specific antibody B152, Quality Control C line 34 on nitrocellulose filter 3
For the sheep anti mouse polyclonal antibody being coated on nitrocellulose filter 3；
Six, pad 2 is containing H HCG specific antibody B207, and detection line T1 32 is for being coated with on nitrocellulose filter 3
Anti-β-HCG antibody, detection line T2 33 is for being coated with H HCG specific antibody B152, Quality Control C line 34 on nitrocellulose filter 3
For the sheep anti mouse polyclonal antibody being coated on nitrocellulose filter 3；
Seven, pad 2 is containing H HCG specific antibody B207, and detection line T1 32 is for being coated with on nitrocellulose filter 3
H HCG specific antibody B152, detection line T2 33 is for being coated with anti-β-HCG antibody, Quality Control C line 34 on nitrocellulose filter 3
For the sheep anti mouse polyclonal antibody being coated on nitrocellulose filter 3；
Eight, pad 2 is containing H HCG specific antibody B207, and detection line T1 32 is for being coated with on nitrocellulose filter 3
H HCG specific antibody B152, detection line T2 33 is for being coated with anti alpha-HCG antibody, Quality Control C line 34 on nitrocellulose filter 3
For the sheep anti mouse polyclonal antibody being coated on nitrocellulose filter 3；
Nine, pad 2 is containing H HCG specific antibody B207, and detection line T1 32 is for being coated with on nitrocellulose filter 3
Anti alpha-HCG antibody, detection line T2 33 is for being coated with anti-β-HCG antibody on nitrocellulose filter 3, and Quality Control C line 34 is for being coated on nitre
Sheep anti mouse polyclonal antibody on acid cellulose film 3；
Ten, pad 2 is containing H HCG specific antibody B207, and detection line T1 32 is for being coated with on nitrocellulose filter 3
Anti-β-HCG antibody, detection line T2 33 is for being coated with anti alpha-HCG antibody on nitrocellulose filter 3, and Quality Control C line 34 is for being coated on nitre
Sheep anti mouse polyclonal antibody on acid cellulose film 3；
11, pad 2 is containing H HCG specific antibody B152, and detection line T1 32 is for being coated on nitrocellulose filter 3
Having H HCG specific antibody B207, detection line T2 33 is for being coated with anti-β-HCG antibody, Quality Control C line on nitrocellulose filter 3
34 is the sheep anti mouse polyclonal antibody being coated on nitrocellulose filter 3；
12, pad 2 is containing H HCG specific antibody B152, and detection line T1 32 is for being coated on nitrocellulose filter 3
Having anti-β-HCG antibody, detection line T2 33 is for being coated with h-HCG specific antibody B207, Quality Control C line 34 on nitrocellulose filter 3
For the sheep anti mouse polyclonal antibody being coated on nitrocellulose filter 3；
13, pad 2 has H-HCG specific antibody B152, and detection line T1 32 is for being coated with on nitrocellulose filter 3
Anti alpha-HCG antibody, detection line T2 33 is for being coated with HCG specific antibody B207 on nitrocellulose filter 3, Quality Control C line 34 is bag
By Mus anti-on nitrocellulose filter 3 polyclonal goat-anti body；
14, pad 2 has H-HCG specific antibody B152, and detection line T1 32 is for being coated with on nitrocellulose filter 3
H HCG specific antibody B207, detection line T2 33 is for being coated with anti alpha-HCG antibody, Quality Control C line 34 on nitrocellulose filter 3
For being coated on anti-Mus polyclonal goat-anti body on nitrocellulose filter 3；
15, pad 2 has H-HCG specific antibody B152, and detection line T1 32 is for being coated with on nitrocellulose filter 3
Anti alpha-HCG antibody, detection line T2 33 is for being coated with anti-β-HCG antibody on nitrocellulose filter 3, and Quality Control C line 34 is for being coated on nitre
Anti-Mus polyclonal goat-anti body on acid cellulose film 3；
16, pad 2 has H-HCG specific antibody B152, and detection line T1 32 is for being coated with on nitrocellulose filter 3
Anti-β-HCG antibody, detection line T2 33 is to be coated with anti alpha-HCG antibody on nitrocellulose filter 3, Quality Control C line 34 is for being coated on
Anti-Mus polyclonal goat-anti body on nitrocellulose filter 3；
17, pad 2 has anti alpha-HCG antibody, and detection line T1 32 is special for being coated with H-HCG on nitrocellulose filter 3
Property antibody B207, detection line T2 33 be coated with H HCG specific antibody B152 on nitrocellulose filter 3, Quality Control C line 34 is
It is coated on anti-Mus polyclonal goat-anti body on nitrocellulose filter 3；
18, pad 2 has anti alpha-HCG antibody, and detection line T1 32 is special for being coated with H-HCG on nitrocellulose filter 3
Property antibody B152, detection line T2 33 be coated with H-HCG specific antibody B207 on nitrocellulose filter 3, Quality Control C line 34 is
It is coated on anti-Mus polyclonal goat-anti body on nitrocellulose filter 3；
19, pad 2 has anti alpha-HCG antibody, and detection line T1 32 resists for being coated with anti-β-HCG on nitrocellulose filter 3
Body, detection line T2 33 is for being coated with H-HCG specific antibody B207 on nitrocellulose filter 3, and Quality Control C line 34 is for being coated on nitre
Anti-Mus polyclonal goat-anti body on acid cellulose film 3；
20, pad 2 has anti alpha-HCG antibody, and detection line T1 32 resists for being coated with anti-β-HCG on nitrocellulose filter 3
Body, detection line T2 33 is for being coated with H-HCG specific antibody B152 on nitrocellulose filter 3, and Quality Control C line 34 is for being coated on nitre
Anti-Mus polyclonal goat-anti body on acid cellulose film 3；
21, pad 2 has anti alpha-HCG antibody, and detection line T1 32 is special for being coated with H-HCG on nitrocellulose filter 3
Heterogenetic antibody B207, detection line T2 33 is for being coated with anti-β-HCG antibody on nitrocellulose filter 3, and Quality Control C line 34 is for being coated on
Anti-Mus polyclonal goat-anti body on nitrocellulose filter 3；
22, pad 2 has anti alpha-HCG antibody, and detection line T1 32 is special for being coated with H-HCG on nitrocellulose filter 3
Heterogenetic antibody B152, detection line T2 33 is for being coated with anti-β-HCG antibody on nitrocellulose filter 3, and Quality Control C line 34 is for being coated on
Anti-Mus polyclonal goat-anti body on nitrocellulose filter 3；
23, pad 2 has anti-β-HCG antibody, and detection line T1 32 is for being coated with H-HCG on nitrocellulose filter 3
Specific antibody B207, detection line T2 33 is for being coated with H-HCG specific antibody B152, Quality Control C line on nitrocellulose filter 3
34 for being coated on anti-Mus polyclonal goat-anti body on nitrocellulose filter 3；
24, pad 2 has anti-β-HCG antibody, and detection line T1 32 is special for being coated with H-HCG on nitrocellulose filter 3
Heterogenetic antibody B152, detection line T2 33 is for being coated with H-HCG specific antibody B207, Quality Control C line 34 on nitrocellulose filter 3
For being coated on anti-Mus polyclonal goat-anti body on nitrocellulose filter 3.
25, pad 2 has anti-β-HCG antibody, and detection line T1 32 is for being coated with anti alpha-HCG on nitrocellulose filter 3
Antibody, detection line T2 33 is for being coated with H-HCG specific antibody B207 on nitrocellulose filter 3, and Quality Control C line 34 is for being coated on
Anti-Mus polyclonal goat-anti body on nitrocellulose filter 3.
26, pad 2 has anti-β-HCG antibody, and detection line T1 32 is for being coated with anti alpha-HCG on nitrocellulose filter 3
Antibody, detection line T2 33 is for being coated with H-HCG specific antibody B152 on nitrocellulose filter 3, and Quality Control C line 34 is for being coated on
Anti-Mus polyclonal goat-anti body on nitrocellulose filter 3.
27, pad 2 has anti-β-HCG antibody, and detection line T1 32 is special for being coated with H-HCG on nitrocellulose filter 3
Heterogenetic antibody B207, detection line T2 33 is for being coated with anti alpha-HCG antibody on nitrocellulose filter 3, and Quality Control C line 34 is for being coated on
Anti-Mus polyclonal goat-anti body on nitrocellulose filter 3.
28, pad 2 has anti-β-HCG antibody, and detection line T1 32 is special for being coated with H-HCG on nitrocellulose filter 3
Heterogenetic antibody B152, detection line T2 33 is for being coated with anti alpha-HCG antibody on nitrocellulose filter 3, and Quality Control C line 34 is for being coated on
Anti-Mus polyclonal goat-anti body on nitrocellulose filter 3.
The present invention a kind of H-HCG Rapid immunodiagnosis reagent paper can be to be fabricated to the form of card, pen.When dual channel mode system
Cheng Ka, pen form time, be cased with a shell outside reagent paper, shell is designed with described sample pad 1, described in add fluid cushion 21, described
Nitrocellulose filter 3, described sample suction pad 4 well 7 of correspondence, liquid filling hole 8, nitrocellulose filter form 9, sample suction pad respectively regard
Window 10 (as shown in Figure 5).
Embodiment one:
The preparation method of described a kind of H-HCG Rapid immunodiagnosis chromatographic test paper, comprises the steps:
Primary raw material is as follows:
Reagent: gold chloride, trisodium citrate, anti alpha-HCG or the antibody of β-HCG, H-HCG specific antibody B207 or
B152；, BSA, trehalose, NaCl, Na2HPO4 12H2O, NaH2PO4 2H2O, boric acid, Borax, tween 20, Triton
X-100, sucrose, ultra-pure water；
Consumptive material: nitrocellulose filter (NC film), 8975 glass fibre, GF-06 glass fibre, adsorptive pads, base plate, capacity
Bottle, conical flask, EP pipe, rifle head.
One, the preparation before preparation:
1.1 2% citrate three sodium solution preparations
Weigh 0.105g citrate three sodium in 15mlEP pipe, add 5ml pure water, mix to being completely dissolved and be 2% lemon
Solution is received in lemon acid three.
Take 40ml pure water in 50ml volumetric flask, in volumetric flask, add 0.5ml 1% chlorauric acid solution, use pure water constant volume
To 50ml, mixing is 0.01% chlorauric acid solution.50ml 0.01% chlorauric acid solution is poured in 100ml conical flask, is placed on
It is heated to boiling with 3-4 shelves on electric furnace, draws 380 μ L 2% citrate three sodium solution with liquid-transfering gun, be quickly driven into boiling
In chlorauric acid solution, continue heating, i.e. 40-55nm gold colloidal is prepared complete.
1.2 PBS preparations:
Weigh Na2HPO4·12H2O 1.615-2g, NaH2PO4·2H2O 0.225-5g, NaCl 4.0-5.0g is in beaker
In, add the dissolving of 400ml ultra-pure water and be settled to 500ml, standby after 0.22 μm membrane filtration.
1.3 redissolution liquid preparations:
Weigh BSA 1g, sucrose 10-13g in beaker, add 70ml PBS and dissolve, add 0.08-0.1ml Triton
X-100, mixing, it is settled to 100ml, standby after 0.22 μm membrane filtration.
2, Optimal pH determines:
Take 1mL gold colloidal respectively in 6 1.5mLEP pipes, be separately added into 0.2M K to often pipe2CO3 1μL、2μL、3μL、5
μ L, 7 μ L, 10 μ L, corresponding pH respectively may be about 7.0,7.5,8.0,8.5,9.0,9.5, is separately added into 150-200 μ L to often pipe
0.1mg/mL antibody-solutions, mixing, 37 degree stand 20min, are separately added into 100-130 μ L10%NaCl solution to often pipe, mixing,
Stand and observe color change, 0.2M K2CO3Addition be 1-3 μ L EP pipe in gold colloidal color still for aubergine, other pipes become
For blueness, optimum pH is 7.0-8.0.
3, minimum antibody labeling amount determines:
Take 4 EP pipes, take 1mL gold colloidal respectively and add in each pipe, then be separately added into K to often pipe2CO32-4 μ L, mixing, add
Enter the antibody-solutions of the 0.08-0.1mg/ml of 30 μ L (3 μ g), 50 μ L (5 μ g), 70 μ L (7 μ g), 100 μ L (10 μ g), mixing, 37
DEG C stand 20min, to often pipe add 10%NaCl solution, mixing, stand observe color change, antibody addition is 7-10 μ g
In EP pipe, gold colloidal color does not occurs significant change to be aubergine, antibody addition be 3,5 μ g EP pipe in gold colloidal become indigo plant, say
The minimum antibody addition of bright 1ml gold colloidal is 5-8 μ g.
4, prepared by gold labeling antibody:
Take the gold colloidal 1mL of 40nm-55nm respectively in EP pipe, in pipe, be separately added into K2CO3Solution 2-4 μ L, mixing,
In EP pipe, add 0.08-0.1mg/ml antibody 70 μ L, mixing, place 20min, from pipe, take 100 μ L manage in 500 μ LEP for 37 DEG C
In, adding 10 μ L10%NaCl solution, mixing, it is red for standing and observing color, can carry out follow-up test.To 40-55nm's
Adding 100 μ L 5%PEG solution in gold colloidal EP pipe, 37 DEG C stand 2h and close, and 4 DEG C of 10000g are centrifuged 20min, abandon
Clearly, redissolving with 100 μ L redissolution liquid, 4 DEG C save backup.
5、
0.2M boric acid solution is prepared: weigh boric acid 12.37g in beaker, adds after 800ml ultra-pure water dissolves and is settled to
1L, standby after 0.22 μm membrane filtration.
0.05M borax soln is prepared: weigh Borax 19.07g in beaker, adds after 800ml ultra-pure water dissolves and is settled to
1L, standby after 0.22 μm membrane filtration.
10mM pH8.0 boric acid-borate buffer solution preparation: take 3ml 0.05M borax soln, 7ml 0.2M boric acid respectively molten
Liquid, in beaker, adds 190ml ultra-pure water, mixes standby.
10mM pH8.4 boric acid-borate buffer solution preparation: take 4.5ml 0.05M borax soln, 5.5ml 0.2M boron respectively
Acid solution, in beaker, adds 190ml ultra-pure water, mixes standby.
Two, preparation method
1., the preparation of sample pad 1:
Preparation sample liquid, weighs 0.5g ± 0.05gBSA in 100ml beaker, adds 70ml 10mM pH8.4 borate
Buffer solution, adds 0.3ml tween 20, mixing, is settled to 100ml, standby after 0.22 μm membrane filtration；
Again the glass fibre membrane after cutting out as strip wide for one-tenth 0.7cm and 3.2cm is placed in soaking at room temperature in sample liquid
4h, 37 DEG C of drying, drying basin saves backup；
2., the preparation of pad 2:
Preparation pad 2 treatment fluid: weigh 0.1g ± 0.05gBSA, 0.5g ± 0.05g trehalose in 100ml beaker,
Add 70ml 10mM pH8.0 borate buffer solution to dissolve, add 0.3ml tween 20, mixing, be settled to 100ml, 0.22 μm
After membrane filtration standby；
The preparation of gold labeling antibody: take 1mL 40-55nm gold colloidal in EP pipe, add 2 μ L 0.2M K2CO3Solution, mixed
Even, add 70 μ L 0.08-0.1mg/ml antibody-solutions, mixing, 37 DEG C stand 20min, add 100 μ L 10%BSA solution (eventually
Concentration 1%), mixing, place 1.5h for 37 DEG C, 4 DEG C, 12000g is centrifuged 20min, abandons supernatant, adds 1ml PBS and redissolves, 4 DEG C,
12000g is centrifuged 20min, abandons supernatant, adds 100 μ L redissolution liquid and redissolves, and 4 DEG C save backup；
Pad 2 raw material is cut into strip wide for 0.7cm again and is placed on soaking at room temperature 4h in pad 2 treatment fluid, 37 DEG C
Dry, then with draw film metal spraying machine according to the amount of 8-15 μ L/cm golden labeling antibody is sprayed on soaking after pad 2 raw material, 37 DEG C
Dry, drying basin saves backup；
3., nitrocellulose filter 3 is coated: preparation is coated buffer: weighs 3g trehalose in beaker, adds 80-
100mlPBS dissolves, and adds 3-5ml methanol, mixing, is settled to 100ml, standby after 0.22 μm membrane filtration；
It is coated detection line T1 32: with being coated buffer, antibody is diluted to 0.5-2mg/ml, by the amount of 0.9-1.5 μ L/cm
Draw on described nitrocellulose filter 3, after 37 DEG C of drying, drying basin saves backup.
It is coated detection line T2 33: be diluted to 0.5-2mg/ml with being coated buffer antibody, draw by the amount of 0.9-1.3 μ L/cm
On described nitrocellulose filter 3, after 37 DEG C of drying, drying basin saves backup.
It is coated Quality Control C line 34: sheep anti mouse polyclonal antibody is diluted to 1.0-1.5mg/ml, by 0.7-with being coated buffer
The amount of 1.4 μ L/cm is drawn on described nitrocellulose filter 3, saves backup after 37 DEG C of drying in drying basin；
4., the preparation of reagent paper:
Prepared by single channel reagent paper:
In temperature 20-30 DEG C, the humidity hothouse less than 30-40%, take gripper shoe 5, by the most coated cellulose nitrate
Element film 3 is placed on the middle part of gripper shoe 5 and pastes；
Glue in detection T line 31 side of described nitrocellulose filter 3 overlap joint pad 2 (taking 1/3rd of pad 2)
Patch, pastes sample pad 1 (taking 1/10th of sample pad 1), the matter on nitrocellulose filter 3 at pad 2 opposite side overlap joint
Control C line 34 side overlap joint sample suction pad 4 (taking 1/10th of sample suction pad 4)；At one layer of marking film of patch topmost, i.e. H-HCG is quick
Immunologic diagnosis reagent paper is successfully prepared, and finally with cutter, the reagent paper of preparation is cut into the wide test strips of 3-4mm.The test strips cut
Can be alone, it is also possible to reinstall in plastic clip or rod (pen), form HCG-H quick diagnosis test card or rod (pen)；
Prepared by dual pathways reagent paper:
In temperature 20-30 DEG C, the humidity hothouse less than 30-40%, take gripper shoe 5, by the most coated described nitric acid
Cellulose membrane 3 is placed on the middle part of gripper shoe 5 and pastes；
Paste described sample suction pad 4 in described nitrocellulose filter 3 side, and described sample suction pad 4 side is fine with described nitric acid
Dimension element film 3 overlaps, and opposite side edge is concordant with described gripper shoe 5, described nitrocellulose filter 3 opposite side and described sample pad 1
Overlap joint so that sample to be tested passes sequentially through described sample pad 1, described nitrocellulose filter 3, described sample suction pad 4 form the first water
Flat flow channel；
Described fiber isolated film 6 two ends are overlapped on above described sample pad 1, described nitrocellulose filter 3 respectively, institute
State pad 2 two ends and be overlapped on above described fiber isolated film 6, described nitrocellulose filter 3 respectively, described in add fluid cushion 21 liang
End be overlapped on above fiber isolated film 6, described pad 2 respectively so that during detection buffer pass sequentially through described in add
Fluid cushion 21, described pad 2, described nitrocellulose filter 3, described sample suction pad 4 form the second bottom horizontal flow sheet passage, i.e. H-HCG
Rapid immunodiagnosis reagent paper is successfully prepared, and finally with cutter, the reagent paper of preparation cuts into the wide test strips of 4mm, the examination cut
Paper slip can load in plastic clip or rod (pen), forms HCG-H quick diagnosis test card or rod (pen)；
The concrete research process of the above-mentioned dual pathways is: when the detected material in sample reaches described by the first bottom horizontal flow sheet passage
After nitrocellulose filter 3, the specific antibody on described detection T line 31 is combined, and after 10 seconds-5 minutes, described sample suction pad 4 has
During sample chromatography, described in add on fluid cushion 21 with buffer, the label (conjugate) in described pad 2 passes through the second level stream
Dynamic passage reaches on described nitrocellulose filter 3, and the detectable substance specific antibody complex on described detection T line 31 is combined
React in macroscopic color (male/female) afterwards.For convenience or sample suction pad 4 described in more accurate observation has no specimen layer
Analysis, described sample suction pad 4 can add coloring agent or painted stripes, develops the color when described sample suction pad 4 has sample to chromatograph, the most just may be used
Adding addition buffer at fluid cushion 21 described, the method is better than and/or preferentially added fluid cushion 21 described again after 10 seconds-5 minutes
Place adds the mode of buffer.
The lap of splice described above is 2mm；And in dual pathways test strips, apply to described in add the buffer on fluid cushion 21
It is different target buffer that are different according to detectable substance and that select, goes out result for quick diagnosis.Three, reagent paper test and result are sentenced
Disconnected
1, test strips test:
Single channel: test strips is put into and stops 5s in liquid to be measured (making well 7 be immersed under liquid level), take out and lie in platform
On face, read result after 15min, in 30min effectively；
The dual pathways: test strips is put into and stops 5s in liquid to be measured (making well 7 be immersed under liquid level), take out and lie in platform
On face, when the color in described sample suction pad form 10 is become after redness from white, take 50 μ LPBS and drop in liquid filling hole 8, after 5min
Read result, in 30min effectively.
2, result judges:
Such as Fig. 1, Fig. 3: when occur two red stripes, one be positioned at detection line T, another be positioned at Quality Control C line 34, show
Conceived, or show that the placental function probability good, generation miscarriage of anemia of pregnant woman is minimum；When red stripes, only a position occur
In Quality Control C line 34, show there is no pregnancy or show that the placental function of anemia of pregnant woman is poor；At Quality Control C line 34 without red when 5-10 minute
Vitta band occurs, shows that incorrect operating process or test strips are rotten and damages, needs correctly to operate by new test strips
And/or retest.
Such as Fig. 2, Fig. 4, when three red stripes occur, one be positioned at detection line T1 32, be positioned at detection line T2 33,
Another is positioned at Quality Control C line 34, shows conceived, and the placental function of anemia of pregnant woman is good, occur the chance miscarried minimum；When going out
Existing two red stripes, one be positioned at detection line T1 32 or T2, another be positioned at Quality Control C line 34, show conceived, but
The placental function of anemia of pregnant woman is bad, the probability of miscarriage occurs greatly, needs to check further and/or treatment；When a red bar occurs
Band, is only located at Quality Control C line 34, shows do not have pregnancy；Occurred at Quality Control C line 34 redfree band when 5-10 minute, show not
Correct operating process or test strips are rotten to be damaged, and needs correctly to operate by new test strips and/or retest.
Embodiment two
The sensitivity Detection of H HCG Rapid immunodiagnosis chromatographic test paper
1, the preparation of 12.5mIU/ml, 25mIU/ml, 50mIU/ml HCG solution: will by the PBS solution containing 0.5%BSA
HCG standard substance redissolve, then are configured to 12.5mIU/ml, 25mIU/ml, 50mIU/ml HCG solution.
2, the preparation of 110mIU/ml H-HCG solution: H-HCG standard substance are redissolved by the PBS solution containing 0.5%BSA, then
It is configured to 110mIU/ml solution.
3, single channel: the test strips of same lot number is respectively put into 12.5mIU/ml, 25mIU/ml, 50mIU/ml HCG molten
Stopping about 5s in liquid and 110mIU/ml H-HCG solution, taking-up lies in and waits about 15min on table top, observed result, detection knot
Really in 30min effectively.Each detectable concentration is respectively repeated 3 times, and 3 testing results of each solution are the positive just can be judged to time concentration
Result is positive；
The dual pathways: the test strips of same lot number is respectively put into 12.5mIU/ml, 25mIU/ml, 50mIU/ml HCG solution
And 110mIU/ml H-HCG solution stops about 5s (making well 7 be immersed under liquid level), take out and lie on table top, work as suction
Color in sample pad form 10 is become after redness from white, takes 50 μ L PBS solution and is added in upper strata liquid filling hole 8, waits about
10min reads result, and in testing result 30min effectively, each detectable concentration is respectively repeated 3 times, and 3 testing results of each solution are
The positive result that just can be judged to time concentration is the positive.
Table 1 mono-/bis-passage sensitivity tests
Note: "-" represents invisible, " +++ " represents it is apparent that 110=110mIU/ml H-HCG solution.
As known from Table 1: single channel and dual pathways sensitivity tests 12.5mIU/ml, 25mIU/ml, 50mIU/ml HCG mark
During quasi-product, HCG line is normal, meets Standard, the non-outlet of H-HCG line, illustrates that HCG standard substance and H-HCG antibody are anti-without intersecting
Should；HCG and the equal outlet of H-HCG line during test 110mIU/ml H-HCG standard substance, illustrate that H-HCG standard substance resist with anti alpha-HCG
Body, anti-β-HCG antibody and H-HCG antibody B207/B152 all have preferable cross reaction.
Embodiment three
The specific detection of H HCG Rapid immunodiagnosis chromatographic test paper
1, solution preparation: 0mIU/ml HCG solution: weigh 0.5gBSA in beaker, adds 70ml PBS and dissolves
It is settled to 100ml, standby after 0.22 μm membrane filtration.
2,500mIU/ml human luteinizing hormone (hLH) solution: hLH standard substance are multiple by the PBS solution containing 0.5%BSA
Molten, being configured to concentration with 0mIU/ml HCG solution is 500mIU/ml, is hLH A liquid；With 25mIU/ml HCG solution
Being configured to concentration is 500mIU/ml, is hLH B liquid；Being configured to concentration with 110mIU/ml H-HCG solution is
500mIU/ml, is hLH C liquid.
3,1000mIU/ml HFSH (hFSH) solution: by the PBS solution containing 0.5%BSA by hFSH standard substance
Redissolving, being configured to concentration with 0mIU/ml HCG solution is 1000mIU/ml, is hFSH A liquid；Use 25mIU/ml HCG
It is 1000mIU/ml that solution is configured to concentration, is hFSH B liquid；It is configured to dense with 110mIU/ml H-HCG solution
Degree is 1000mIU/ml, is hFSH C liquid.
4,1000 μ IU/ml human thyrotropin (hTSH) solution: hLH standard substance are multiple by the PBS solution containing 0.5%BSA
Molten, being configured to concentration with 0mIU/ml HCG solution is 1000 μ IU/ml, is hTSH A liquid；Molten with 25mIU/ml HCG
It is 1000 μ IU/ml that liquid is configured to concentration, is hTSH B liquid；It is configured to concentration with 110mIU/ml H-HCG solution
It is 1000 μ IU/ml, is hTSH C liquid.
5, negative specificity:
Single channel: it is 500mIU/ml hLH A liquid, 1000mIU/ml that the test strips of same lot number is respectively put into concentration
Stopping about 5s in hFSH A liquid, 1000 μ IU/ml hTSH A liquors, taking-up lies in and waits about 15min on table top, observes knot
Really, in testing result 30min effectively, each concentration is respectively repeated 3 times, and 3 testing results of each solution are feminine gender and then judge that this is molten
The specificity of liquid is negative；
The dual pathways: it is 500mIU/ml hLH A liquid, 1000mIU/ml that the test strips of same lot number is respectively put into concentration
Stopping about 5s (making well 7 be immersed under liquid level) in hFSH A liquid, 1000 μ IU/ml hTSH A liquors, taking-up lies in
On table top, take 50 μ L PBS solution when the color in sample suction pad form 10 is become redness from white and be added in upper strata liquid filling hole 8,
Waiting that about 10min reads result, in testing result 30min effectively, each concentration is respectively repeated 3 times, and 3 testing results of each solution are equal
Then judge that for feminine gender the specificity of this solution is as negative.
Table 2 mono-/bis-passage feminine gender specificity test result
Note: "-" represents invisible, " +++ " represents clearly visible
As known from Table 2: the negative specificity test HCG line of single channel and dual pathways test strips and H-HCG line are feminine gender,
Article two, the equal non-false positive of T line occurs, this index meets Standard.
6, positive specificity:
Single channel: the test strips of same lot number is respectively put into concentration be 500mIU/ml Hlh B liquid and C liquid,
1000mIU/ml hFSH B liquid and C liquid, 1000 μ IU/ml hTSH B liquid and C liquor stop about 5s, takes out and lie in platform
About 15min is waited on face, observed result, in testing result 30min effectively.Each concentration is respectively repeated 3 times, 3 detections of each solution
Result is feminine gender and then judges that the specificity of this solution is as negative；
The dual pathways: the test strips of same lot number is respectively put into concentration be 500mIU/ml hLH B liquid and C liquid,
1000mIU/ml hFSH B liquid and C liquid, 1000 μ IU/ml hTSH B liquid and C liquor stop about 5s and (makes well 7 submergence
Under liquid level), take out and lie on table top, when the color in sample suction pad form 10 is become after redness from white, take 50 μ L PBS
Solution is added in upper strata liquid filling hole 8, waits that about 10min reads result, and in testing result 30min effectively, each concentration respectively repeats 3
Secondary, 3 testing results of each solution are the positive and then judge that the specificity of this solution is as positive.
Table 3 mono-/bis-passage positive specificity test result
Note: "-" represents invisible, " ++ " represents visible, and " +++ " represents clearly visible
Knowable to 3 tables: the positive specificity test HCG line of single channel and dual pathways test strips and H-HCG line are the positive,
Article two, T line all occurs without false negative, and this index also complies with Standard.
Embodiment four:
The Detection of Stability of H HCG Rapid immunodiagnosis chromatographic test paper
By the test strips of same lot number 37 DEG C place test paper bar sensitivity respectively after 7d, 15d, 21d, specificity and
Repeatability, sensitivity and specificity method of testing are with embodiment two and embodiment three.
Reperformance test method is as follows:
1, single channel: each time point takes 20 test strips and puts into 25mIU/ml HCG standard substance or 110mIU/ml
Stopping about 5s in H-HCG solution, taking-up lies in and waits about 15min on table top, observed result, in testing result 30min effectively.
2, the dual pathways: each time point takes 20 test strips and puts into 25mIU/ml HCG standard substance or 110mIU/ml
H-HCG solution stops about 5s (making well 7 be immersed under liquid level), takes out and lie on table top, when in sample suction pad form 10
Color become after redness from white, take 50 μ L PBS solution and be added in upper strata liquid filling hole 8, wait about 10min read result, inspection
Survey result effective in 30min.
Test strips single, double passage lowest detectable limit test result after 4 37 DEG C of table, 21 days destruction
Note: "-" represents invisible, " ++ " represents visible, and " +++ " expression is it is apparent that 110=110mIU/ml H-HCG
Solution.
Test strips feminine gender specificity test result after 5 37 DEG C of table, 21 days destruction
Note: "-" represents invisible, " +++ " represents clearly visible
Test strips positive specificity test result after 6 37 DEG C of table, 21 days destruction
Note: "-" represents invisible, " ++ " represents visible, and " +++ " represents clearly visible
Test strips reperformance test result after 7 37 DEG C of table destruction
Note: "-" represents invisible, " ++ " represents visible, and " +++ " represents clearly visible
Knowable to table 4-7: single channel and dual pathways test strips test sensitivity, specificity and repetition after 37 DEG C are destroyed
Property, the equal non-false positive of result and false negative, meet Standard, illustrate that test strips has good stability.
Embodiment five:
The clinical experiment of H HCG Rapid immunodiagnosis chromatographic test paper
To the clinical urine collected, blood, use the application test strips, detect according to using method, face with hospital
Bed checks that result compares, and hospital's blood examination result shows that the blood-prostatae barrier value having 5 parts of samples in 50 parts of samples is less than 1.2mIU/ml,
Being diagnosed as not conceived, for negative sample, the blood-prostatae barrier value of other 45 parts of samples is more than 5mIU/ml, is diagnosed as pregnancy, for positive sample
This, through other clinical auxiliary examination, be diagnosed to be 5 parts of tires in 45 parts of positive sample and stop educating sample；Urine by these 50 parts of samples
Liquid, blood use this ELISA test strip respectively, and 5 parts of negative sample of test strips detection are consistent with hospital clinical result false sun does not occurs
Property, other 45 parts of samples of test strips detection are positive consistent with hospital clinical result there is not false negative, HCG line coincidence rate
100%, 5 parts of tires that additionally this ELISA test strip goes out in 45 parts of positive sample stop educating sample and hospital clinical diagnostic result also
Causing, it is also 100% that tire is stopped to educate detection coincidence rate by H-HCG line, illustrates that the test strips of the application can detect normal pregnancy simultaneously
With the tentative diagnosis that tire stops abnormal pregnancy or the miscarriage educated.This test strips clinical trial is shown in Table 8:
Table 8 test strips clinical test results
Note: "-" represents invisible, " +++ " represent it is apparent that ++ represent visible ,+faint visible
Wen Zhong, H-HCG represent H-hCG.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all spirit in the present invention and
Within principle, any modification, equivalent substitution and improvement etc. made, should be included within the scope of the present invention.
Claims (10)
1. a H-HCG Rapid immunodiagnosis chromatographic test paper, it is characterised in that: include the sample pad (1) being sequentially connected with, be coated with
Detection T line (31) and the nitrocellulose filter (3) of Quality Control C line (34), sample suction pad (4), and described sample pad (1), described nitric acid
Cellulose membrane (3), described sample suction pad (4) overlap successively, are pasted onto in a gripper shoe (5).
2. a kind of H-HCG Rapid immunodiagnosis chromatographic test paper, it is characterised in that:
Also include a pad (2), and described pad (2) is arranged on described sample pad (1) and described nitrocellulose filter (3)
Between so that described sample pad (1) constitutes single with described pad (2), described nitrocellulose filter (3), described sample suction pad (4)
Passage reagent paper.
3. a kind of H-HCG Rapid immunodiagnosis chromatographic test paper, it is characterised in that:
Also include that a fiber isolated film (6), a pad (2) and add fluid cushion (21), and described fiber isolated film (6) two
End is overlapped on described sample pad (1), described nitrocellulose filter (3) top respectively, and described pad (2) two ends are overlapped on respectively
Described fiber isolated film (6), described nitrocellulose filter (3) top, described in add fluid cushion (21) two ends and be overlapped on described respectively
Fiber isolated film (6), described pad (2) top so that described sample pad (1) and described fiber isolated film (6), described
Pad (2) and described add fluid cushion (21), described nitrocellulose filter (3), described sample suction pad (4) constitute dual pathways reagent paper.
4. a kind of H-HCG Rapid immunodiagnosis chromatographic test paper as described in Claims 2 or 3, it is characterised in that: described pad
(2) it is provided with H-HCG specific antibody B207 or B152 on, described nitrocellulose filter (3) T line is coated with H-HCG special
Property antibody B152 or B207, described Quality Control C line (34) contain sheep anti mouse polyclonal antibody；
Or,
It is provided with anti alpha-HCG or the antibody of β-HCG, or anti-complete HCG antibody on described pad (2), or is provided with H-
HCG specific antibody B207 or B152；Detection T line (31) is made up of detection line T1 (32), detection line T2 (33), and described detection
Line T1 (32) is coated with on anti alpha-HCG or the antibody of β-HCG, or anti-complete HCG antibody, detection line T2 (33) and is coated with H-
It is coated with H-HCG specific antibody B152 or B207, inspection on HCG specific antibody B152 or B207, or detection line T1 (32)
Anti alpha-HCG or the antibody of β-HCG, or anti-complete HCG antibody it is coated with on survey line T2 (33),
Or,
It is suitable for Immune competition method, described nitrocellulose filter (3) T line is coated with H-HCG antigen or contrary, pad (2)
On be provided with H-HCG antigen, T line is coated with H-HCG specific antibody.
5. the preparation method of a H-HCG Rapid immunodiagnosis chromatographic test paper, it is characterised in that: comprise the steps:
1., the preparation of sample pad (1): prepare sample pad treatment fluid, then the glass fibre membrane after cutting out or non-woven fabrics are placed in
Soaking at room temperature in sample pad treatment fluid, drying, standby；
2., the preparation of pad (2): prepare pad treatment fluid, gold labeling antibody, pad raw material is placed in pad treatment fluid
Middle soaking at room temperature, drying, then with draw film metal spraying machine by gold labeling antibody be sprayed on soaking after pad raw material, dry for standby；
3., nitrocellulose filter (3) is coated: preparation is coated buffer, dilutes sheep anti mouse Anti-TNF-α respectively with being coated buffer
Body, anti alpha-HCG or the antibody of β-HCG, or anti-complete HCG antibody, H-HCG specific antibody B207 or B152 to 0.1-
20mg/ml, it is thus achieved that Quality Control C line (34), detection line T1 (32), detection line T2 (33) or Quality Control C line (34), detection line T2
(33), detection line T1 (32) or Quality Control C line (34), detection T line (31)；
4., the preparation of reagent paper:
Single channel reagent paper: in the hothouse of room temperature, takes described gripper shoe (5), first by above-mentioned coated described celluloid
Film (3) fits in the middle part of described gripper shoe (5), then paste respectively in nitrocellulose filter (3) both sides described pad (2),
Described sample suction pad (4), and paste described sample pad in the side that described pad (2) is relative with described nitrocellulose filter (3)
(1), cutter is finally used to shear described gripper shoe (5)；
Dual pathways reagent paper: described nitrocellulose filter (3) side paste described sample suction pad (4), and described sample suction pad (4) side with
Described nitrocellulose filter (3) overlap, opposite side edge is concordant with described gripper shoe (5), described nitrocellulose filter (3) another
Side and described sample pad (1) overlap so that sample to be tested pass sequentially through described sample pad (1), described nitrocellulose filter (3),
Described sample suction pad (4) forms the first bottom horizontal flow sheet passage；
Described fiber isolated film (6) two ends are overlapped on respectively described sample pad (1), described nitrocellulose filter (3) top,
Described pad (2) two ends be overlapped on respectively described fiber isolated film (6), described nitrocellulose filter (3) top, described in add
Fluid cushion (21) two ends are overlapped on fiber isolated film (6), described pad (2) top respectively so that buffer during detection
Add fluid cushion (21) described in passing sequentially through, described pad (2), described nitrocellulose filter (3), described sample suction pad (4) form the
Two bottom horizontal flow sheet passages, finally use cutter to shear described gripper shoe (5).
6. as claimed in claim 5 preparation method, it is characterised in that: described step 1. in, the preparation method of sample treatment solution is:
Weigh 0.5g ± 0.05gBSA in beaker, add 70ml 10mM pH8.4 borate buffer solution and dissolve, add tween 20, mixed
Even, standby after constant volume, membrane filtration.
7. as claimed in claim 5 preparation method, it is characterised in that: step 2. in, the preparation method of pad treatment fluid is: claim
Take 0.1g ± 0.05gBSA, 0.5g ± 0.05g trehalose in beaker, add 70ml 10mM pH8.0 borate buffer solution molten
Solve, add tween 20, standby after mixing, membrane filtration；Containing anti alpha-HCG or the antibody of β-HCG in described gold labeling antibody, or
Anti-complete HCG antibody, or H-HCG specific antibody B207 or B152.
8. as claimed in claim 5 preparation method, it is characterised in that: described step 3. in, the preparation method being coated buffer is:
Take trehalose in beaker, add after PBS dissolves and add methanol, standby after mixing, constant volume, membrane filtration.
9. preparation method as described in claim 6 to 8, it is characterised in that: described sample pad (1) material is glass fibre membrane or nothing
Spin cloth；Described sample suction pad (4) is made up of absorbent filter.
10. preparation method as described in claim 6 to 8, it is characterised in that: described pad (2) is by 5-100nm gold colloidal or 5-
800nm nano material or latex particle or carbon granule or other luminescent dye or enzyme combines Anti-HCG antibody or H-HCG specificity resists
Body B207 or B152 forms, or quotes or combine Avidin, biotin；Described pad is marked with specific antibody in (2)
Gold colloidal or cellulose nano-particle, or it is marked with the gold colloidal cellulose nano-particle of specific antibody, or in the two
It is added with Avidin or biotin.
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| CN108459167A (en) * | 2018-03-16 | 2018-08-28 | 江苏维尔生物科技有限公司 | A kind of kit and preparation method thereof quantitatively detected for β-HCG, PDG in human urine |
| CN109752562A (en) * | 2018-06-04 | 2019-05-14 | 军事科学院军事医学研究院军事兽医研究所 | A kind of human chorionic gonadotropin-nano flower and its preparation method and application |
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| CN111289744A (en) * | 2020-03-17 | 2020-06-16 | 长春万成生物电子工程有限公司 | Novel colloidal gold test paper for coronavirus detection |
| CN111596073B (en) * | 2020-06-04 | 2023-05-02 | 昆明天沃生物科技有限公司 | Method for detecting early pregnancy of sheep |
| CN111596073A (en) * | 2020-06-04 | 2020-08-28 | 昆明天沃生物科技有限公司 | Method for detecting early pregnancy of sheep |
| CN112730855A (en) * | 2020-12-28 | 2021-04-30 | 杭州新脉生物科技有限公司 | Colloidal gold chromatography test strip based on anti-human beta-HCG bispecific antibody |
| CN113156105A (en) * | 2021-01-27 | 2021-07-23 | 中国人民解放军军事科学院军事医学研究院 | A type botulinum toxin rapid quantitative detection card |
| CN113063938A (en) * | 2021-03-13 | 2021-07-02 | 河南省农业科学院 | High-sensitivity gradient semi-quantitative immunochromatography detection test strip and detection method |
| CN113063938B (en) * | 2021-03-13 | 2023-09-12 | 河南省农业科学院 | High-sensitivity gradient semi-quantitative immunochromatography detection test strip and detection method |
| CN116819070A (en) * | 2023-07-10 | 2023-09-29 | 希莱乐检(郑州)生物科技有限公司 | Test strip, detection device and detection method for detecting target in body fluid |
| CN116819070B (en) * | 2023-07-10 | 2024-03-22 | 希莱乐检(郑州)生物科技有限公司 | Test strip, detection device and detection method for detecting target in body fluid |
| CN117452002A (en) * | 2023-12-25 | 2024-01-26 | 山东康华生物医疗科技股份有限公司 | Human chorionic gonadotrophin colloidal gold detection test strip and kit for urine saliva simultaneous detection |
| CN117452002B (en) * | 2023-12-25 | 2024-03-22 | 山东康华生物医疗科技股份有限公司 | Human chorionic gonadotrophin colloidal gold detection test strip and kit for urine saliva simultaneous detection |
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