CN101216493A - Test paper for diagnosing premature rupture of fetal membrane and reagent kit - Google Patents
Test paper for diagnosing premature rupture of fetal membrane and reagent kit Download PDFInfo
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- CN101216493A CN101216493A CNA2008100467297A CN200810046729A CN101216493A CN 101216493 A CN101216493 A CN 101216493A CN A2008100467297 A CNA2008100467297 A CN A2008100467297A CN 200810046729 A CN200810046729 A CN 200810046729A CN 101216493 A CN101216493 A CN 101216493A
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Abstract
The invention relates to a test strip and a kit for premature rupture of membrane. The test strip comprises, from top to bottom of the horizontal plane of the test strip, a water absorbent pad, a cellulose nitrate membrane, a gold standard pad and a sample pad, wherein a detection line and a quality control line are coated on the cellulose nitrate membrane; the detection line is a monoclonal antibody 11B6 and the quality control line is rabbit anti-mouse IgG antibody; a monoclonal antibody 14A3 marked by colloidal gold is coated on the gold standard pad; and the absorbent pad, the cellulose nitrate membrane coated with the detection line and the quality control line, the gold standard pad coated with the monoclonal antibody 14A3 marked by the colloidal gold and the sample pad are sequentially adhered on a water-nonabsorbent support thin sheet from top to bottom; and a sample injecting hole is provided on the sample pad. The test strip in the kit has the advantages of high specificity and high sensitivity, and can be used for clinic diagnosis of premature rupture of membrane. The kit has simple operation and can determine the result in ten minutes.
Description
Technical field
The invention belongs to immune application, relate to association areas such as molecular biology, immunity, medical science.Relate to premature rupture of fetal membranes specifically, amniotic fluid is excessive, detects the test strips and the kit of specific label in the excessive amniotic fluid and then this disease of diagnosis premature rupture of fetal membranes then.
Background technology
Premature rupture of fetal membranes (premature rupture of membrane, PROM) be obstetrics' common complication, easily cause complication such as fetal in utero infection, fetal distress, enclose the living youngster's mortality ratio and the incidence of disease and raise, accurately do not assess the fetal in utero safety but there is desirable method.The rupture of membranes incidence is at 6.6%-13.9% before the childbirth in all patients, and at ensuing 48 hours, spontaneous abortion can take place 70%-90% in these cases.Premature rupture of fetal membranes not only directly or indirectly causes the premature labor of 4.5%-14%, and causes significant mortality ratio and the morbidity rate relevant with childbirth.Respectively neonate's result of study is being shown in 109 and 111 pregnant woman research, 34 weeks or before PROM can cause the mortality ratio of 16.8%-26.6% and an incidence of disease of 68.8% generally.Therefore set up a kind of simple PROM diagnostic method accurately very significance is arranged.In some cases, PROM can be according to limpid amniotic fluid that discharges in the vagina and existing of amniotic fluid afterwards, and judge easily.PROM is not obvious like that in some cases, and is difficult to distinguish the amniotic fluid of leakage and the cervical secretions of urine and vagina.
Can pass through a lot of diagnosis of program PROM according to the literature, these programs mainly comprise: the evaluation of fetus flakey epithelial cell or fine hair, after fetal cell is painted within it transparent fetoprotein in the measurement of diamine oxidase active, vaginal mucus prolactin, the vaginal mucus in the amniotic fluid of portion or outside crystal type, heating, the vaginal mucus, the insulin-like growth factor in the vaginal mucus to the method for fetal cell fat globule microscopic examination, the estimation of vaginal mucus pH, dry amniotic fluid in conjunction with measurement of albumen, inhibin B or the like.Yet all these diagnostic methods all are to carry out under the situation that PROM has taken place, and they are not entirely accurates.Because in most of the cases, the existence of interfering material and some produce in the vagina the inside, such as urine, meconium, pregnancy serum and blood.The existence of these interfering materials is easy to cause the erroneous judgement to PROM.
Immunochromatography (immunochromatography) is the immunoassay mode that comes across a kind of uniqueness at the eighties initial stage, the core technology of this method is to be solid phase with the fibre strip chromatographic material, make sample solution swimming on chromatography strip by capillarity, and make on determinand in the sample and the chromatographic material immune response that high special, high-affinity takes place at the acceptor of determinand such as antigen or antibody simultaneously.Colloidal gold immunochromatographimethod is analyzed (gold-immunochroma tography assay GICA) and is used colloidal gold-labeled method, as tracer, is applied to a kind of novel immunolabelling technique of antigen-antibody reaction with collaurum.In the chromatography process, gold label and prior immobilization are in chromatographic material: be that antigen on the NC film or antibody specific immune response takes place and are trapped as nitrocellulose filter, further enrichment forms the visible aubergine band of naked eyes, thereby obtain experimental result intuitively, reach the purpose of detection.This method is had relatively high expectations to employed antigen-antibody, requires to have good specificity and high-purity.
According to this principle, developed and a lot of colloidal gold immune chromatography rapid detecting test paper strips.Cure application facet the people, used the gold-marking immunity chromatograph test strip at the antigen and the aspects such as antibody, venereal disease cause of disease, bacterium, parasite, tumor marker, cardiovascular disease mark, medicine and some other protein of hormone, infectious disease cause of disease both at home and abroad.In animal doctor's application facet, for example also used the gold-marking immunity chromatograph test strip at aspects such as swine fever, canine parvovirus diseases.In the diagnosing premature rupture of fetal membrane related fields, have three patents.First be called the apparatus and method that detect amniotic fluid in the vaginal fluid (international application no is: PCT/US2003/025125 2003.8.12), though this patent is identical with the patent principle of the present patent application: be the double antibodies sandwich colloidal gold immunity chromatography.But the present invention and existing this patent key material on the utilization said method is different fully: two kinds of monoclonal antibodies that existing patent is used are at the different epitopes of a1-microglobulin (PAMG-1), and two kinds of monoclonal antibodies used in the present invention are at AMNI-Protein or AMNI-Pr.The kit specificity of the present invention's assembling is better, and susceptibility is higher.Another patent name is a disposable premature rupture of fetal membranes pH test paper diagnosis rod (200620083734.1), and the shortcoming of this invention is that susceptibility is not high, easily causes false negative.Another patent name A MARKER FOR PROLONGED RUPTURE OF MEMBRANES (WO2007030890), the method that detects amniotic fluid in this patent is the ELISA method, this method is compared shortcoming with colloidal gold immunochromatographimethod method be length consuming time, need operating instrument, operating personnel are had relatively high expectations.
Summary of the invention
Purpose of the present invention is in order to overcome the problem that above-mentioned prior art exists, and a kind of test strips and kit that is used to detect premature rupture of fetal membranes is provided, and test strips of the present invention and kit specificity are better, and susceptibility is higher.
The present invention is achieved in that
A kind of test strips that is used for diagnosing premature rupture of fetal membrane, the test strips surface level is followed successively by from top to bottom: adsorptive pads, nitrocellulose filter, gold mark pad, sample pad, it is characterized in that: be coated with detection line and nature controlling line on the nitrocellulose filter, wherein detection line is monoclonal antibody 11B6, and nature controlling line is a rabbit anti-mouse igg antibody; Be coated with the monoclonal antibody 14A3 of colloid gold label on the gold mark pad; Again with absorption pad, be coated with the nitrocellulose filter of detection line and nature controlling line, be coated with the gold mark pad of the monoclonal antibody 14A3 of colloid gold label, sample pad sticks to successively on the support slice that does not absorb water by top-down order and assembles, and on sample pad, be provided with a well, wherein monoclonal antibody 11B6 and monoclonal antibody 14A3 are: the specific marker thing in the amniotic fluid is AMNI-Protein or AMNI-Pr, the disclosed patent No. is: WO/2007/030890, and at the different epitopes of specific marker thing AMNI-Protein or AMNI-Pr and the hybridoma cell strain 11B6 and the 14A3 that prepare.This two strains cell is numbered: 14A3 and 11B6, be deposited in Chinese typical culture collection center (CCTCC) on 01 17th, 2008, and deposit number is respectively: CCTCC-C200801 and CCTCC-C200802.Described nitrocellulose filter, absorption pad, gold mark pad, sample pad, the support slice that does not absorb water all can be available from Millipore company.
The preparation method of described monoclonal antibody 11B6 and 14A3: (1) is with amniotic fluid specific proteins AMNI-Pr immunity BALB/c mouse: inject mouse peritoneal after getting the amniotic fluid specific proteins AMNI-Pr protein solution that contains 150 μ g and isopyknic Freund's complete adjuvant emulsification, after 21 days, inject mouse peritoneal after getting the amniotic fluid specific proteins AMNI-Pr protein solution that contains 150 μ g and isopyknic incomplete Freund emulsification, at last in merging first three day or being separated by more than 4 weeks with for the first time immune time, carry out reinforced immunological in the abdominal cavity of BALB/c mouse again, the antigen amount is doubled to 300 μ g, does not add adjuvant; When (2) merging, learn from else's experience one of the BALB/c mouse of last reinforced immunological, the eye socket sacrificed by exsanguination, collect positive serum, in concentration 75% alcoholic solution, soak the 5min sterilization, aseptic taking-up mouse spleen is isolated splenocyte, with the SP2/0 myeloma cell of prepared fresh, the mixing in the 50mL centrifuge tube in 1～2 * 107 SP2/0 and 1: 10～1: 15 ratio of 108 immune spleen cells, 1500rpm, centrifugal 10min outwells supernatant, the centrifuge tube that cell mixture is housed is put in 37 ℃ of water-baths, the concentration that slowly splashes into pre-temperature to 37 ℃ then in 1min is 50% polyglycol (PEG) 0.8mL, and the limit edged stirs with pipette tip, continues to stir 1min; The PRMI-1640 basal liquid 10mL that adds 37 ℃ of pre-temperature then, PRMI-1640 is available from GIBCO company, concrete grammar is: dropwise splashed into 1mL in first minute, add 1mL in second minute, added 3mL on the 3rd～4 minute, added remaining 5mL on the 5th minute, add 30mL PRMI-1640 liquid at last, the centrifugal 5min of 800rpm removes supernatant, places 5～8min in 37 ℃; Suspend with the HAT nutrient culture media; (3) will plant 250 μ L/ holes in 96 well culture plates with the cell mixture branch that the HAT nutrient culture media suspends, every hole inoculum concentration contains 104 SP2/0 cells, and in 37 ℃, concentration is 5%CO
2Cultivate in the incubator, after the fusion second day begins to observe to be had pollution-freely, added 1 HAT nutrient culture media in the 4th day, suction in the 8th～10 day goes 100 μ L nutrient culture media to change HT nutrient culture media 100 μ L, treat that the fused cell colony grows to culture hole 1/4, during the nutrient culture media flavescence, carry out antibody test; Adopt immunogene as screening antigen, utilize enzyme linked immunosorbent assay (ELISA) method to filter out the positive hole of the anti-AMNI-Pr of secretion; (4) the positive hole that screens is cloned, screened with limiting dilution assay, through 3～4 time clonings, finishing screen is selected the monoclonal hybridoma of the anti-AMNI-Pr of secretion two strains, and this two strains cell is numbered: 14A3 and 11B6.
A kind of kit that is used for diagnosing premature rupture of fetal membrane is placed on described test strips, sample diluting liquid, cotton swab, centrifuge tube, suction pipe in the same box, and sample diluting liquid is the physiological saline of concentration 0.85%.
Principle of the present invention be monoclonal antibody 11B6 as the detection line encrusting substance, monoclonal antibody 14A3 utilizes the double antibodies sandwich method to detect and whether contains amniotic fluid in the sample to be checked as the antibody of colloid gold label.During detection, in the sample to be checked if when having amniotic fluid to exist in the amniotic fluid polypeptide marker thing at first with golden labelled antibody 14A3 antigen-antibody reaction to take place form compound polypeptide marker thing-Jin labeled monoclonal antibody 14A3 compound, because capillarity, the reaction compound is along nitrocellulose filter (NC film) swimming forward, when arriving detection line, run into the monoclonal antibody 11B6 on the NC film, will form monoclonal antibody 11B6-polypeptide marker thing-Jin labeled monoclonal antibody 14A3 compound, thereby be enriched on the detection line, form the red precipitate line, the compound that is not combined on the detection line continues to spring up forward, is enriched at last on the nature controlling line, is judged to positive findings; When if gold mark monoclonal antibody 14A3 arrives detection line when not containing amniotic fluid in the sample, run into the monoclonal antibody 11B6 that is coated on the detection line and just antigen-antibody reaction can not take place, therefore just can enrichment on detection line yet, gold mark monoclonal antibody 14A3 passes through detection line, be enriched on the nature controlling line, form the red precipitate line, be judged to negative findings.
Compared with prior art the present invention has following advantage:
1, amniotic fluid detection excessive in the premature rupture of fetal membranes is had high specificity, highly sensitive, detection time is short, as long as 5～10 minutes, test sample can be blood, vaginal mucus, uterine neck liquid, test sample wide ranges like this;
2, detection method of the present invention has the low characteristics of the cost of detection without any need for specific apparatus, equipment;
3, detection kit of the present invention is easy and simple to handle, does not need to be operated by the professional;
4, kit of the present invention stores conveniently, and is not high to temperature requirement, and effective storage life can reach 1 year under 4～8 ℃; At room temperature can preserve six months.
Description of drawings
Fig. 1 is the vertical view of test strips of the present invention.
Fig. 2 is the side view of test strips of the present invention.
Among the figure: 1-adsorptive pads, 2-nature controlling line, 3-detection line, 4-gold mark pad, 5-sample pad, 6-nitrocellulose filter, 7-liner plate.
Embodiment
The preparation of AMNI-Pr
The amniotic fluid sample obtains 25-975ml on one's body from the women through caesarean operation, and installing in the 5ml centrifuge tube a the branch with 2ml, filters with glass wool before the packing.
Every pipe 2ml, mix 20 pipe amniotic fluid and prepare sample cell, purifying with the following method then: at first use blue glue (the Aurum Affi-Gel Blue Mini Kit) pre-service of Affi-Gel to remove the endogenous albumin, the blue glue of Affi-Gel is available from U.S. Bio-Rad company, according to the sample in the blue glue instructions of the Affi-Gel that the is bought pretreatment sample pond.
Next by the sample of handling with the blue glue of Affi-Gel is that the immunochromatographic method that WO03/018634 describes is handled with international patent, can obtain the specific albumen of amniotic fluid.
Be respectively 300kD and 100kD centrifugal ultrafiltration pipe (AmiconUltra-4) with two kinds of molecular cut offs at last, 300kD and 100kD centrifugal ultrafiltration pipe are available from U.S. Mi Libo (Millipore) company, handle the amniotic fluid specific proteins according to purchase centrifugal ultrafiltration pipe instructions, can obtain three kinds of filtered solutions at last, be respectively AF1, AF2 and AF3 by the big minispread of molecular weight, the specific marker thing that these three kinds of labels are in the amniotic fluid is the preparation of AMNI-Pr.
Anti-AMNI-Pr MONOCLONAL ANTIBODIES SPECIFIC FOR
With reference to the method in Xue Qingshan " philosophy and technique of in vitro culture " the Science Press calendar year 2001 version: with above-mentioned prepared AF1, AF2 and AF3 immune BALB/c mouse respectively, BALB/c mouse is available from Hubei Province's medical courses in general institute Experimental Animal Center, immune programme for children is to get AF1 or AF2 or AF3 protein solution and the isopyknic Freund's complete adjuvant emulsification that contains 150 μ g, Freund's complete adjuvant is available from Sigma company, the injection mouse peritoneal, booster immunization after 21 days, use incomplete Freund emulsification instead, incomplete Freund is available from Sigma company; In merging first three day, also can be to be separated by more than 4 weeks, at last at the abdominal cavity of BALB/c mouse reinforced immunological with for the first time immune time, the antigen amount is doubled to 300 μ g, does not add adjuvant, during fusion, learn from else's experience one of the BALB/c mouse of last reinforced immunological, the eye socket sacrificed by exsanguination is collected positive serum, soaks the 5min sterilization in 75% alcohol, aseptic taking-up mouse spleen, isolate splenocyte, with the SP2/0 myeloma cell of prepared fresh, by 2 * 10
7Individual SP2/0 and 10
81: 15 ratio of individual immunocyte mixing in the 50mL centrifuge tube, SP2/0 myeloma cell is available from Ministry of Public Health's Wuhan institute of Biological Products, 1500rpm, centrifugal 10min.Outwell supernatant, also the filter paper of available sterilization blots, and knocks the pipe end gently, makes cell precipitation loosening slightly; The centrifuge tube that cell mixture is housed is put in 37 ℃ of water-baths, slowly splashes into 50% polyglycol (PEG) 0.8mL of pre-temperature to 37 ℃ then in 1min, 50% polyglycol is available from Sigma company, and the limit edged stirs with pipette tip gently, continues to stir 1min; The PRMI-1640 basal liquid 10mL that slowly add 37 ℃ of pre-temperature then, PRMI-1640 are available from Sigma company, the prescription of basal liquid: PRMI-164010.4g, NaHCO
32g is settled to 1000M1 with distilled water, transfers pH to 7.2; Concrete grammar is: dropwise splashed into 1mL in first minute, added 1mL in second minute, added 3mL on the 3rd～4 minute, added remaining 5mL on the 5th minute, each added-time needs slowly to add, and constantly stirs lightly; Add 30mL PRMI-1640 liquid at last, also need slowly to add, the centrifugal 5min of 800rpm removes supernatant, places 5～8min in 37 ℃; Suspend with the HAT nutrient culture media, HAT is available from Sigma company, the composition of HAT nutrient culture media: 80mL basal liquid, the 20mL calf serum of sterilizing, the HAT of 1mL100%, 1mL is two anti-, two anti-preparations: get novocillin 1,000,000 units and gentamicin sulphate 1g and dissolve in the 100mL tri-distilled water; Simultaneously also with the HAT nutrient culture media suspend the feeder cells that prepare and with fusion after mixing with cells.Add the HAT nutrient culture media as required, divide and plant in 96 well culture plates, about 250 μ L/ holes.Once merge and to inoculate 4～8 96 orifice plates.Also can plant less as required, generally press the cell number of SP2/0 and calculate, every hole inoculum concentration contains about 104 SP2/0 cells approximately.In 37 ℃, 5%CO
2Cultivate in the incubator.After the fusion second day begins to observe to be had pollution-freely, added 1 HAT nutrient culture media in the 4th day, and suction in the 8th～10 day goes 100 μ L nutrient culture media to change HT nutrient culture media 100 μ L, HT is available from Sigma company, the composition of HT nutrient culture media: 80mL basal liquid, the 20mL calf serum of sterilizing, the HT of 1mL100%, 1mL is two anti-; Treat that the fused cell colony grows to culture hole 1/4, during the nutrient culture media flavescence, carry out antibody test; Adopt immunogene as screening antigen, utilize the ELISA method to filter out the positive hole of the anti-AMNI-Pr of secretion.The positive hole that screens is cloned, screened with limiting dilution assay at once, and limiting dilution assay is with reference to Xue Qingshan " (philosophy and technique of in vitro culture " Science Press's calendar year 2001 version; Through 3～4 time clonings, finishing screen is selected the monoclonal hybridoma of the anti-AMNI-Pr of secretion two strains, this two strains cell is numbered: 14A3 and 11B6, be deposited in Chinese typical culture collection center (CCTCC) on 01 17th, 2008, deposit number is respectively: CCTCC-C200801 and CCTCC-C200802.This cell has been carried out chromosome counting, and the result shows that the chromosome average of SP2/0 is 70, and splenocyte chromosome is 40, and the number of hybridoma is between 81～93.The chromosome number that all is higher than two parent's cells illustrates the cell of SP2/0 really of fused cell and the hybridization product of splenocyte, and the chromosome of hybridoma is obviously more than the chromosome of myeloma cell SP2/0.With this injection cell BALB/c mouse abdominal cavity, manufacture order clonal antibody.Employing is carried out hypotype evaluation, mouse IgG2b subclass as a result available from the mouse source monoclonal antibody hypotype identification kit (Mouse Mab Isotyping Test Kit) of ROCKLAND company to the resulting monoclonal antibody of the present invention.
1. the preparation of ascites
After adopting sterilized liquid paraffin pre-service BALB/c mouse, the method production ascites of injecting hybridoma again, its detailed process is as follows:
The pre-service of BALB/c mouse: the whiteruss with sterilization is handled mouse, and lumbar injection 0.5mL/ is the injectable hybridoma after ten days only.
The enlarged culture in positive hole: add feeder cells at 24 porocyte culture plates, 4/hole, forward the positive cell in 96 orifice plates to 24 porocyte culture plates and carry out enlarged culture.
Injection: by the time cell covers with at the bottom of the hole that the cell of 24 orifice plates is resuspended, 1200r/min, centrifugal 5min, cell count is diluted to 5 * 106/milliliter with the RPMI-1640 basic culture solution, and intraperitoneal injection of mice 0.5mL/ only, observe the mouse web portion protuberance about 8 days, can gather ascites.
2. the pre-service of ascites
3000g, centrifugal 10min gets supernatant, and supernatant is with 0.45 μ m membrane filtration.
3. the preliminary purification of monoclonal antibody uses hydroxyapatite chromatography post purifying in the ascites
Hydroxyapatite (HA) chromatographic column is available from BioRad company
The principle of hydroxyapatite (HA) chromatographic column
Ca2+ group and the biomolecule of HA: reacting to each other of the negative charge group on protein, nucleic acid, virus and other living matter surface plays an important role in HA separation of biomolecules process.And reacting to each other of the positive electricity group of the PO4-3 group of HA and biomolecule surface then plays secondary role.
The preparation of hydroxyapatite (HA) chromatographic column damping fluid
The damping fluid of balance pillar: 25mmol/L phosphate buffer, pH7.0.Concrete prescription is: 5.45gNa
2HPO
4.12H
2O, 1.52gNaH
2PO
4.2H
2O is settled to 1000ml.
The damping fluid of wash-out pillar: 500mmol/L phosphate buffer, pH7.0.Concrete prescription is: 109gNa
2HPO
4.12H
2O, 30.4gNaH
2PO
4.2H
2O is settled to 1000ml.
The step that hydroxyapatite (HA) chromatographic column uses:
With the direct upper prop of pretreated ascites supernatant.
With 25mmo l/L phosphate buffer flushing pillar, until OD280 less than 0.2.
With 500mmol/L phosphate buffer wash-out pillar, collect eluting peak.
With the eluting peak desalination of collecting,, phosphate is reduced to 20mmol/L with ultrafiltration cup or dialysis desalting.
4. with monoclonal antibody purifying gel affinity chromatography (HiTrap Protein G HP) purification column monoclonal antibody in the ascites is further purified
HiTrap Protein G HP purification column is available from GE Heal thcare Bio-Sciences AB company.
The principle of this purification column of HiTrap Protein G HP
Protein G is an albumen of G class streptococcus cell surface, is an III type Fc acceptor that can combine with the Fc of IgG by non-immunologic mechanism, and the principle of this combination and the a-protein of staphylococcus aureus are similar.Yet a-protein has different binding specificities with protein G, and this depends primarily on the source of IgG.Compare with a-protein, protein G is stronger in conjunction with monoclonal antibody IgG the time, such as the monoclonal antibody IgG from ox, sheep and horse.Different with a-protein is that protein G can be in conjunction with mouse monoclonal antibody IgG, human IgG 3 and small white mouse IgG1.
Protein G in the high-performance protein G Ago-Gel of filling in this purification column of HiTrap Protein G HP is produced by recombinant protein in Escherichia coli, and this recombinant protein contains two IgG calmodulin binding domain CaMs.The albumin calmodulin binding domain CaM of native protein G is deleted in genetic process, and therefore the IgG of this reorganization can avoid and albuminised cross reaction.
The purpose that this purification column of HiTrap Protein G HP is designed is exactly to separate also IgG from the how anti-or monoclonal antibody from ascites, serum and cells and supernatant that purifying comes out.
The preparation of sample acidity level pad behind this purification column balance of the HiTrap Protein G HP purifying:
Because protein G and IgG have very strong affinity when pH7.0, so used eluent pH value is lower usually when wash-out.Because thereby the IgG behind some purifying may be to the responsive activity that influences it of low excessively acidity, for fear of the generation of this situation, we have added some acidity level pads usually in advance when collecting sample.The prescription of acidity level pad is: 1M Tris-HC1, pH9.0 handles with 0.45 μ m membrane filtration.Concrete prescription is: 121g three (methylol) aminomethane (Tris-base), transfer pH9.0 with concentrated hydrochloric acid, and be settled to 1000ml.
The preparation of this purification column damping fluid of HiTrap Protein G HP
Adsorption-buffering liquid: the 20mM sodium phosphate buffer, pH7.0 is with 0.45 μ m membrane filtration.Concrete prescription is: the 3.12g sodium dihydrogen phosphate, transfer pH7.0 with 1M NaOH, and be settled to 1000ml.
Elution buffer: 0.1M glycocoll-hydrochloride buffer, pH2.7 is with 0.45 μ m membrane filtration.Concrete prescription 0.75g glycocoll, the 0.28ml concentrated hydrochloric acid, pH2.7 is settled to 100ml.
This purification column of HiTrap Protein G HP is to the requirement of sample preparation:
The sample processing of at first will desalting, this can be by realizing the sample desalting processing with adsorption-buffering liquid dilute sample or with HiTrap, HiPrepTM26/10 or these several desalination pillars by ion-exchange of PD-10.
Sample after the desalting processing need be handled with 0.45 μ m membrane filtration.
The operation steps that this purification column of HiTrap Protein G HP uses:
In collection tube, add 200 μ L acidity level pads.
Adsorption-buffering liquid with 10 times of column volumes is washed pillar, and flow velocity is 1ml/min.
According to the amount of capacity of pillar, suitably select the amount of a upper prop sample in conjunction with monoclonal antibody.
Adsorption-buffering liquid with 10 times of volumes is washed pillar, removes non-binding thing in the sample.
With the elution buffer wash-out pillar of 5 times of volumes, each collection tube is collected 1ml.
The purifying thing of collecting can be realized the sample desalting processing by HiTrap, HiPrepTM26/10 or these several desalination pillars by ion-exchange of PD-10.
The preparation of rabbit anti-mouse igg antibody:
With reference to chief editors such as He Zhaoyang, " (the animal immunology experimental technique " Changchun: Jilin science tech publishing house, method in 2002: utilize the microorganism at inventor place and the BALB/c mouse IgG immune health rabbit that extract the immunization experiment chamber, the rabbit anti-mouse igg hyper-immune serum that preparation high specific, height are tired, adopt the saturated ammonium sulphate method slightly to carry to hyper-immune serum, after G-200 crosses post, obtain highly purified rabbit anti-mouse igg antibody.Utilize this antibody to can be used as nature controlling line.
Wrap the preparation of the nitrocellulose filter of tested survey line and nature controlling line
1, bag is cushioned the preparation of liquid: 0.01M pH7.2 phosphate buffer (PBS damping fluid) is cushioned liquid for bag, 0.22 μ membrane filtration mistake, put 4 ℃ standby, one week of the term of validity; 1000ml 0.01M pH7.2PBS buffer formulation: NaCl8g, KCl0.2g, Na
2HPO
412H
2O 2.9g, KH
2PO
40.2g two ionized waters that boil off are settled to 1000ml.
2, the preparation of the nitrocellulose filter of tested survey line of bag and nature controlling line: be cushioned liquid with bag 11B6 is diluted to 1mg/ml, adjust machine, be scribed ss the T line, be detection line, the T line is near gold mark pad end, apart from the about 5mm of gold mark pad end; With bag be cushioned liquid with the rabbit anti-mouse igg antibody dilution to 0.5mg/ml, adjust machine, be scribed ss the C line, be nature controlling line, the C line is near absorption pad, apart from the about 3mm of absorption pad.Two linear distances, 5～8mm, line should be careful, even.37 ℃ of oven dry encapsulate standby.
The preparation of collaurum, golden labeled monoclonal antibody
1, the preparation of solution
The preparation of (1) 1% gold chloride: boil off ionized water dissolved chlorine auric acid with two, be made into 1% solution, put 4 ℃ standby, the term of validity four months.1000ml1% chlorauric acid solution prescription: 10g gold chloride; Two ionized waters that boil off are settled to 1000ml.
(2) preparation of trisodium citrate: with two ionized waters dissolving trisodium citrates that boil off, be made into 1% solution, 0.22 μ membrane filtration mistake, put 4 ℃ standby, be settled to 1000ml.
(3) 0.1MK
2CO
3Preparation: boil off the ionized waters preparation with two, 0.22 μ membrane filtration mistake, put 4 ℃ standby, the term of validity four months.1000ml 0.1MK
2CO
3Solution formula: 13.8gK
2CO
3, two ionized waters that boil off are settled to 1000ml.
The preparation of (4) 5% bovine serum albumin(BSA)s (BSA): boil off the ionized waters preparation with two, 0.22 μ membrane filtration mistake, put 4 ℃ standby, the term of validity four months.100ml 5%BSA solution formula: 5g BSA; Two ionized waters that boil off are settled to 100ml.
(5) boric acid of the preparation of borate buffer: 2mM, 0.2% PEG-20000,0.02% Sodium azide (NaN
3).The prescription of 1000ml borate buffer is: 0.124g boric acid, 2g PEG-20000,0.2gNaN
3, 0.22 μ membrane filtration mistake boils off ionized water and is settled to 1000ml with two.
2, the preparation of collaurum:
With two ionized waters that boil off 1% gold chloride is diluted to 0.01%, put on the heating magnetic stirring apparatus heated and stirred and boil, add the 2ml1% trisodium citrate, continue to boil by every 100ml 0.01% gold chloride, be shiny red up to liquid and promptly stop heating, supply dehydration after being cooled to room temperature.The collaurum outward appearance for preparing should be pure, bright, do not have precipitation and floating thing, one week of the term of validity.
3, colloid gold label MONOCLONAL ANTIBODIES SPECIFIC FOR:
Use 0.1MK
2CO
3Transfer the pH value to 8.2 of collaurum, add monoclonal antibody 14A3 by 8 μ g antibody/ml collaurum, magnetic stirring apparatus mixing 30min, stir down adding BSA to final concentration be 1%, left standstill 1 hour.8600rpm, 4 ℃ of centrifugal 45min, abandon supernatant, precipitation is resuspended with borate buffer, use 12000rpm, 4 ℃ of centrifugal 30min then, abandon supernatant, precipitation repeats above-mentioned once centrifugal, uses the borate buffer of 1/10th initial collaurum volumes will precipitate resuspended at last, put 4 ℃ standby, one week of the term of validity.
The gold that applies the monoclonal antibody 14A3 of colloid gold label is marked the preparation of filling up
1, the preparation of confining liquid:
2%BSA, 2% polyvinylpyrrolidone K-30 (PVP-K30), 0.05%NaN
3, 0.01M pH7.2PBS solution, 0.22 μ m miillpore filter filter, put 4 ℃ standby, the term of validity four months.1000ml confining liquid prescription: 20g BSA, 0.5g NaN3,20g PVP-K30,0.01M pH7.2PBS solution are settled to 1000ml.
2, the gold that applies the monoclonal antibody 14A3 of colloid gold label is marked the preparation of filling up:
Gold mark pad is soaked in the confining liquid behind the 30min, in 37 ℃ of oven dry.The colloidal gold solution that concentrates good labelled antibody among the embodiment 4 is diluted with borate buffer, and ratio is 1: 4.5.Colloidal gold solution with the labelled antibody for preparing is layered on the gold mark pad uniformly then, 20 square centimeters of every ml soln shops, freeze drying, encapsulation, put 4 ℃ standby.
Embodiment 8
The preparation of test strips sample pad
1, the preparation of confining liquid:
2%BSA, 2%PVP-K30,0.05%NaN3,0.01M pH7.4PBS solution, 0.22 μ m miillpore filter filter, put 4 ℃ standby, the term of validity four months.1000ml confining liquid prescription: 20g BSA, 0.5g NaN
3, 20g PVP-K30,8gNaCl, 2.9gNa
2HPO
412H
2O, 0.2gKCl, 0.2gKH
2PO
4Solution is settled to 1000ml.
2, the preparation of sample pad:
Sample pad is soaked in the confining liquid behind the 30min, in 37 ℃ of oven dry, encapsulation, put 4 ℃ standby.
Embodiment 9
The assembling of test strips
The nitrocellulose filter of bag tested survey line and nature controlling line, the gold mark pad, absorption pad, the sample pad that apply the monoclonal antibody 14A3 of colloid gold label are sticked to successively on the support slice that does not absorb water by Fig. 1 and assemble, and on sample pad, be provided with a well, be cut into wide little of 3mm.Per ten little one bags add drying agent, Vacuum Package.4～8 ℃ of preservations are valid for one year; Normal temperature is preserved, the term of validity 6 months.
Embodiment 10
The composition of diagnosing premature rupture of fetal membrane kit
1, the diagnosing premature rupture of fetal membrane kit comprises:
(1), test strips
(2), sample diluting liquid
(3), cotton swab
(4), centrifuge tube
(5), suction pipe
2, the phase order closes the preparation of solution
Sample diluting liquid: sample diluting liquid is 0.85% NaCl solution.Compound method: 8.5g NaCl adds and two boil off ionized water and be settled to 1000ml.
The using method of kit of the present invention:
1, specimen preparation: dip in the mucus of getting vagina or uterine neck collar extension with cotton swab, 0.85% normal saline flushing and the overstocked cotton swab with about 0.5ml obtains then.
2, detect: take out kit, equilibrium at room temperature 20 minutes; Open the package, take out test strips, draw the sample that has prepared, in the test strips well, drip 2-3 then and drip and get final product with suction pipe.
3, the result judges: when macroscopic aubergine nature controlling line appears in test strips, macroscopic aubergine detection line do not occur, the result is judged to feminine gender, is designated as "-"; When macroscopic aubergine nature controlling line appears in test strips, simultaneously macroscopic aubergine detection line also appears, and the result is judged to the positive, is designated as "+"; The amniotic fluid content of the detected sample of the dark more explanation of detection line color is high more; When macroscopic aubergine nature controlling line does not appear in test strips, no matter macroscopic aubergine detection line whether occurs, the result is judged to test strip and lost efficacy, and should discard.
Embodiment 11
The application of diagnosing premature rupture of fetal membrane kit kit
1, the source place of sample collecting sample collecting is the how tame hospital of Wuhan and these two places, Xiangfan.The collection of amniotic fluid mainly obtains on one's body from caesarean women.Other clinical sample is from pregnant woman's vaginal mucus, blood and uterine neck liquid etc., and the approach of collection is to dip in the mucus of getting vagina or uterine neck collar extension with cotton swab, and 0.85% normal saline flushing and the overstocked cotton swab with about 0.5ml obtains then.
Specificity test with 0.85% physiological saline, take from the normal pregnancies vaginal mucus sample, take from amniotic fluid of pregnant woman sample, take from the normal not sample of conceived woman vagina mucus and be added drop-wise to the test strips well respectively.The test findings that obtains is for except that the amniotic fluid sample is positive, and other several samples are all negative.Also gathered 26 parts of normal pregnant woman's blood and urine in addition, the result of test is:
| The sample and the numbering that detect | Pregnant woman's pregnancy period (week) | |
| 1 pregnant woman's blood | 32 | |
| 2 pregnant woman's blood | 34 | |
| 3 pregnant woman's blood | 36 | |
| 4 pregnant woman's blood | 36 | |
| 5 pregnant woman's blood | 34 | |
| 6 pregnant woman's blood | 29 | |
| 7 pregnant woman's blood | 34 | Negative |
| 8 pregnant woman's blood | 33 | Negative |
| 9 pregnant woman's blood | 35 | Negative |
| 10 pregnant woman's blood | 36 | Negative |
| 11 pregnant woman's blood | 37 | Negative |
| 12 pregnant woman's blood | 36 | Negative |
| 13 pregnant woman's blood | 34 | Negative |
| 14 pregnant woman's blood | 36 | Negative |
| 15 pregnant woman's blood | 36 | Negative |
| 16 pregnant woman's blood | 37 | Negative |
| 17 pregnant woman's blood | 33 | Negative |
| 18 pregnant woman's urine | 33 | Negative |
| 19 pregnant woman's urine | 34 | Negative |
| 20 pregnant woman's urine | 36 | Negative |
| 21 pregnant woman's urine | 32 | Negative |
| 22 pregnant woman's urine | 31 | Negative |
| 23 pregnant woman's urine | 36 | Negative |
| 24 pregnant woman's urine | 36 | Negative |
| 25 pregnant woman's urine | 38 | Negative |
| 26 pregnant woman's urine | 37 | Negative |
The result is known thus, this test strips and pregnant woman's urine or blood no cross reaction.Get 150 parts of amniotic fluid that collect the pregnant woman then, detecting positive rate with test strips of the present invention is 100%.Know that thus this test strips has good specificity.
3, the amniotic fluid sample doubling dilution that will gather from pregnant woman's body of sensitivity tests is added drop-wise to test strips then respectively and detects the result that obtains and can be diluted to 16 times for pregnant woman's amniotic fluid.
4, premature rupture of fetal membranes test strips test card Preliminary Applications clinically
Get 20 parts of artificial premature rupture of fetal membranes case samples of making, detect with test strips test card of the present invention then, the result is as follows:
| The sample and the numbering that detect | Note | Testing result |
| (the collection position: the uterine neck collar extension) of sample before the 1 artificial rupture of membranes | The result appears in 30 minutes | Negative |
| (the collection position: the uterine neck collar extension) of sample before the 2 artificial rupture of membranes | The result appears in 30 minutes | Negative |
| (the collection position: the uterine neck collar extension) of sample before the 3 artificial rupture of membranes | The result appears in 30 minutes | Negative |
| (the collection position: the uterine neck collar extension) of sample before the 4 artificial rupture of membranes | The result appears in 30 minutes | Negative |
| (the collection position: the uterine neck collar extension) of sample before the 5 artificial rupture of membranes | The result appears in 30 minutes | Negative |
| (the collection position: the uterine neck collar extension) of sample before the 6 artificial rupture of membranes | The result appears in 30 minutes | Negative |
| (the collection position: the uterine neck collar extension) of sample before the 7 artificial rupture of membranes | The result appears in 30 minutes | Negative |
| (the collection position: the uterine neck collar extension) of sample before the 8 artificial rupture of membranes | The result appears in 30 minutes | Negative |
| (the collection position: the uterine neck collar extension) of sample before the 9 artificial rupture of membranes | The result appears in 30 minutes | Negative |
| (the collection position: the uterine neck collar extension) of sample before the 10 artificial rupture of membranes | The result appears in 30 minutes | Negative |
| (the collection position: the uterine neck collar extension) of sample behind the 11 artificial rupture of membranes | The result appears in 10 minutes | Positive |
| (the collection position: the uterine neck collar extension) of sample behind the 12 artificial rupture of membranes | The result appears in 10-15 minute | Weak positive |
| (the collection position: the uterine neck collar extension) of sample behind the 13 artificial rupture of membranes | The result appears in 10 minutes | Positive |
| (the collection position: the uterine neck collar extension) of sample behind the 14 artificial rupture of membranes | The result appears in 10 minutes | Positive |
| (the collection position: the uterine neck collar extension) of sample behind the 15 artificial rupture of membranes | The result appears in 10 minutes | Positive |
| (the collection position: the uterine neck collar extension) of sample behind the 16 artificial rupture of membranes | The result appears in 10-15 minute | Weak positive |
| (the collection position: the uterine neck collar extension) of sample behind the 17 artificial rupture of membranes | The result appears in 10-15 minute | Weak positive |
| (the collection position: the uterine neck collar extension) of sample behind the 18 artificial rupture of membranes | The result appears in 10 minutes | Positive |
| (the collection position: the uterine neck collar extension) of sample behind the 19 artificial rupture of membranes | The result appears in 10 minutes | Positive |
| (the collection position: the uterine neck collar extension) of sample behind the 20 artificial rupture of membranes | The result appears in 10 minutes | Positive |
Get 22 parts of samples that are diagnosed as the patient of premature rupture of fetal membranes clinically, the result who detects with test strips test card of the present invention is:
| The sample and the numbering that detect | Note | Testing result |
| 1 clinical diagnosis premature rupture of fetal membranes patient | The result appears in 10 minutes | Positive |
| 2 clinical diagnosis premature rupture of fetal membranes patients | The result appears in 10 minutes | Positive |
| 3 clinical diagnosis premature rupture of fetal membranes patients | The result appears in 10 minutes | Positive |
| 4 clinical diagnosis premature rupture of fetal membranes patients | The result appears in 10-15 minute | Positive |
| 5 clinical diagnosis premature rupture of fetal membranes patients | The result appears in 10 minutes | Positive |
| 6 clinical diagnosis premature rupture of fetal membranes patients | The result appears in 10 minutes | Positive |
| 7 clinical diagnosis premature rupture of fetal membranes patients | The result appears in 10 minutes | Positive |
| 8 clinical diagnosis premature rupture of fetal membranes patients | The result appears in 10 minutes | Positive |
| 9 clinical diagnosis premature rupture of fetal membranes patients | The result appears in 10 minutes | Positive |
| 10 clinical diagnosis premature rupture of fetal membranes patients | The result appears in 10 minutes | Positive |
| 11 clinical diagnosis premature rupture of fetal membranes patients | The result appears in 10-15 minute | Positive |
| 12 clinical diagnosis premature rupture of fetal membranes patients | The result appears in 10 minutes | Positive |
| 13 clinical diagnosis premature rupture of fetal membranes patients | The result appears in 10 minutes | Positive |
| 14 clinical diagnosis premature rupture of fetal membranes patients | The result appears in 10 minutes | Positive |
| 15 clinical diagnosis premature rupture of fetal membranes patients | The result appears in 10 minutes | Positive |
| 16 clinical diagnosis premature rupture of fetal membranes patients | The result appears in 10 minutes | Positive |
| 17 clinical diagnosis premature rupture of fetal membranes patients | The result appears in 10 minutes | Positive |
| 18 clinical diagnosis premature rupture of fetal membranes patients | The result appears in 10 minutes | Positive |
| 19 clinical diagnosis premature rupture of fetal membranes patients | The result appears in 10-15 minute | Positive |
| 20 clinical diagnosis premature rupture of fetal membranes patients | The result appears in 10 minutes | Positive |
| 21 clinical diagnosis premature rupture of fetal membranes patients | The result appears in 10 minutes | Positive |
| 22 clinical diagnosis premature rupture of fetal membranes patients | The result appears in 10 minutes | Positive |
Know tentatively that thus premature rupture of fetal membranes test strips test card of the present invention has the coincidence rate very high with the clinical diagnosis result.
Claims (3)
1. test strips that is used for diagnosing premature rupture of fetal membrane, the test strips surface level is followed successively by from top to bottom: absorption pad, nitrocellulose filter, gold mark pad, sample pad, it is characterized in that: be coated with detection line and nature controlling line on the nitrocellulose filter, wherein detection line is monoclonal antibody 11B6, and nature controlling line is a rabbit anti-mouse igg antibody; Be coated with the monoclonal antibody 14A3 of colloid gold label on the gold mark pad; Again absorption pad, the nitrocellulose filter that is coated with detection line and nature controlling line, gold mark pad, the sample pad that is coated with the monoclonal antibody 14A3 of colloid gold label are sticked to successively on the support slice that does not absorb water by top-down order and assemble, and be provided with a well on sample pad.
2. require the 1 described test strips that is used for diagnosing premature rupture of fetal membrane according to power, it is characterized in that: the preparation method of described monoclonal antibody 11B6 and 14A3: (1) is with amniotic fluid specific proteins AMNI-Pr immunity BALB/c mouse: inject mouse peritoneal after getting the amniotic fluid specific proteins AMNI-Pr protein solution that contains 150 μ g and isopyknic Freund's complete adjuvant emulsification, after 21 days, inject mouse peritoneal after getting the amniotic fluid specific proteins AMNI-Pr protein solution that contains 150 μ g and isopyknic incomplete Freund emulsification, at last in merging first three day or being separated by more than 4 weeks with for the first time immune time, carry out reinforced immunological in the abdominal cavity of BALB/c mouse again, the antigen amount is doubled to 300 μ g, does not add adjuvant; When (2) merging, learn from else's experience one of the BALB/c mouse of last reinforced immunological, the eye socket sacrificed by exsanguination, collect positive serum, in concentration 75% alcoholic solution, soak the 5min sterilization, aseptic taking-up mouse spleen is isolated splenocyte, with the SP2/0 myeloma cell of prepared fresh, the mixing in the 50mL centrifuge tube in 1～2 * 107 SP2/0 and 1: 10～1: 15 ratio of 108 immune spleen cells, 1500rpm, centrifugal 10min outwells supernatant, the centrifuge tube that cell mixture is housed is put in 37 ℃ of water-baths, the concentration that slowly splashes into pre-temperature to 37 ℃ then in 1min is 50% polyglycol (PEG) 0.8mL, and the limit edged stirs with pipette tip, continues to stir 1min; The PRMI-1640 basal liquid 10mL that adds 37 ℃ of pre-temperature then, PRMI-1640 is available from GIBCO, concrete grammar is: dropwise splashed into 1mL in first minute, add 1mL in second minute, added 3mL on the 3rd～4 minute, added remaining 5mL on the 5th minute, add 30mL PRMI-1640 liquid at last, 800rp hinders centrifugal 5min, removes supernatant, places 5～8min in 37 ℃; Suspend with the HAT nutrient culture media; (3) will plant 250 μ L/ holes in 96 well culture plates with the cell mixture branch that the HAT nutrient culture media suspends, every hole inoculum concentration contains 104 SP2/0 cells, and in 37 ℃, concentration is 5%CO
2Cultivate in the incubator, after the fusion second day begins to observe to be had pollution-freely, added 1 HAT nutrient culture media in the 4th day, suction in the 8th～10 day goes 100 μ L nutrient culture media to change HT nutrient culture media 100 μ L, treat that the fused cell colony grows to culture hole 1/4, during the nutrient culture media flavescence, carry out antibody test; Adopt immunogene as screening antigen, utilize enzyme linked immunosorbent assay (ELISA) method to filter out the positive hole of the anti-AMNI-Pr of secretion; (4) the positive hole that screens is cloned, screened with limiting dilution assay, through 3～4 time clonings, finishing screen is selected the monoclonal hybridoma of the anti-AMNI-Pr of secretion two strains, and this two strains cell is numbered: 14A3 and 11B6.
3. kit that is used for diagnosing premature rupture of fetal membrane, it is characterized in that: described test strips, sample diluting liquid, cotton swab, centrifuge tube, suction pipe are placed in the same box, and sample diluting liquid is the physiological saline of concentration 0.85%.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100467297A CN101216493A (en) | 2008-01-21 | 2008-01-21 | Test paper for diagnosing premature rupture of fetal membrane and reagent kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100467297A CN101216493A (en) | 2008-01-21 | 2008-01-21 | Test paper for diagnosing premature rupture of fetal membrane and reagent kit |
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|---|---|
| CN101216493A true CN101216493A (en) | 2008-07-09 |
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| CNA2008100467297A Pending CN101216493A (en) | 2008-01-21 | 2008-01-21 | Test paper for diagnosing premature rupture of fetal membrane and reagent kit |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101813702A (en) * | 2010-04-01 | 2010-08-25 | 苏州市玮琪生物科技有限公司 | Tool and method for diagnosing premature rupture of membrane by continuous monitoring |
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| WO2011150752A1 (en) * | 2010-06-02 | 2011-12-08 | 成都创宜生物科技有限公司 | Rapid detection tool and kit for premature rupture of fetal membrane taking icam-1 as detection index |
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| CN101813702A (en) * | 2010-04-01 | 2010-08-25 | 苏州市玮琪生物科技有限公司 | Tool and method for diagnosing premature rupture of membrane by continuous monitoring |
| CN101813702B (en) * | 2010-04-01 | 2014-05-14 | 顾瑜 | Tool and method for diagnosing premature rupture of membrane by continuous monitoring |
| WO2011150752A1 (en) * | 2010-06-02 | 2011-12-08 | 成都创宜生物科技有限公司 | Rapid detection tool and kit for premature rupture of fetal membrane taking icam-1 as detection index |
| CN101968488A (en) * | 2010-10-26 | 2011-02-09 | 吉林大学 | Colloidal gold fast diagnostic test strip of goose paramyxovirus disease |
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| CN115561458A (en) * | 2022-07-08 | 2023-01-03 | 华中科技大学同济医学院附属协和医院 | Colloidal gold rapid detection card for diagnosing biliary tract occlusion and preparation method thereof |
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