CN102901828A - Test paper used for detecting acute myocardial infarction, and preparation method and application method thereof - Google Patents
Test paper used for detecting acute myocardial infarction, and preparation method and application method thereof Download PDFInfo
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- CN102901828A CN102901828A CN2012102980113A CN201210298011A CN102901828A CN 102901828 A CN102901828 A CN 102901828A CN 2012102980113 A CN2012102980113 A CN 2012102980113A CN 201210298011 A CN201210298011 A CN 201210298011A CN 102901828 A CN102901828 A CN 102901828A
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Abstract
The invention provides a test paper used for detecting acute myocardial infarction, and a preparation method and an application method thereof. The test paper comprises a base plate and sequentially closely connected components adhered to the base plate, wherein the components are a sample absorption pad, a bonding pad, a chromatography membrane, and a water absorption pad. The bonding pad is coated with a glucan-antibody-fluorescein mixed marker. The chromatography membrane is provided with a detection band and a control band. A monoclonal antibody is fixed on the detection band. A rabbit anti-mouse IgG antibody which can be speficically bound with the glucan-antibody-fluorescein mixed marker is fixed on the control band. The sensitivity range of the reagent provided by the invention reaches 0.1ng/L. With the test paper, quantitative detections upon trace myoglobin, troponin I and creatine kinase isoenzyme in myocardial infarction patient serum can be realized. A result can be obtained in 3-5 minutes after sample spotting. The operation is simple, and professional operation is not needed.
Description
Technical field
The present invention relates to the immune diagnostic reagent field, be specially and relate to a kind of test paper that detects acute myocardial infarction, test paper preparation and application process.
Background technology
Along with the aggravation of China's aging population process, acute myocardial infarction has become the major disease of harm China.The World Health Organization (WHO) points out that the whole world has 8,500,000 people to die from acute myocardial infarction every year.In China, the annual patient of acute myocardial infarction is 2,000 ten thousand people, and has 1,000,000 people to be devitalized by acute myocardial infarction.Thereby acute myocardial infarction has become the major disease of harm compatriots health.Treatment shows, is the most important time for the treatment of in 3 hours behind the Pathogenesis of Acute Myocardial Infarction.Only have Patients With Myocardial Infarction is carried out prevention morning, early finds, early gives treatment to, could at utmost save patient's life, at utmost improve patient's prognosis.
Seek the key that early stage myocardial injury markers is the early diagnosis miocardial infarction.Studies show that at present: human muscle hemoglobin, creatine kinase isozyme, Troponin I can be discharged into peripheral blood after the seizure of disease, can be used as the specific index of diagnosis of myocardial infarction.
At present, the main immunological method of domestic detection miocardial infarction has ELISA (ELISA) and gold-marking immunity test strips.ELISA relates to a plurality of steps such as application of sample, warm bath, washing, colour developing, termination, at least need 3 hours, in addition, need to finish in clinical laboratory or centralab, thereby can not be behind Pathogenesis of Acute Myocardial Infarction patient family, the emergency tender launches.ELISA sensitivity can only reach 10ng/ml in addition, if the material below the 10ng/ml is detected, will produce false-negative result, and is namely undetected.Immune colloidal gold technique is with a kind of immunological technique of collaurum as the tracer label thing.By contrast, the susceptibility of colloidal gold technique is poorer than ELISA, can only reach 50ng/ml, to almost be impossible to the trace detection of human muscle hemoglobin, Troponin I, creatine kinase isozyme, present domestic colloidal gold technique still belongs to the starting stage in addition, basically can not to These parameters quantitatively.
In view of above feature, at present domestic the and even world can be in time in the urgent need to setting up one on the diagnosing acute miocardial infarction, hypersensitivity, quantitative detection system, and with the exploitation of said detecting system for the acute myocardial infarction product.
Summary of the invention
For defects and the problem of prior art, the purpose of the embodiment of the invention provides can be for detection of a kind of test paper, test paper preparation and application process that detects acute myocardial infarction of acute myocardial infarction.
In order to achieve the above object, the embodiment of the invention provides following technical scheme: a kind of test paper that detects acute myocardial infarction comprises base plate, is attached to the absorption of sample pad, pad, chromatographic film and the adsorptive pads that closely link to each other successively on the base plate; Wherein, pad is coated with glucosan-antibody-fluorescein mixed mark thing; Have one on the chromatographic film and detect band and a quality control band, detection is with and is fixed with monoclonal antibody; Quality control band be fixed with can with the rabbit anti-mouse igg antibody of glucosan-antibody-fluorescein mixed mark thing specific bond.
The present invention also provides a kind of method for preparing test paper that detects acute myocardial infarction, comprises following steps:
(1) with dilution glucosan-antibody-fluorescein mixed mark thing is diluted to concentration to the 0.01-0.03mg/ml scope, then is immersed in the dilution in connection with pad, after oven dry or vacuum freeze drying, refill and be fitted on the test strips base plate; Consisting of of dilution: add mass percent 0.1% NaN3, mass percent 1% BSA in the PBS damping fluid of pH7.4 0.01M.
(2) in chromatographic film two bands are set, one is detected band and a quality control band; Immobilized monoclonal antibody is with in detection, and described clonal antibody is a kind of of anti-myoglobins, Troponin I and creatine kinase isozyme monoclonal antibody; Quality control band is rabbit anti-mouse igg antibody fixedly;
(3) stick absorption of sample pad and pad in a side of the chromatographic film of step (2), opposite side sticks adsorptive pads; The detection band of absorption of sample pad, pad, chromatographic film, quality control band and the adsorptive pads of chromatographic film are arranged successively.
The present invention also provides a kind of test paper application process that detects acute myocardial infarction, comprises following steps:
(1) adds the test serum sample at the absorption of sample pad; Under capillary action, sample liquid is to adsorptive pads one end swimming, form immune complex at pad place and glucosan-antibody-fluorescein mixed mark thing, again further swimming, immune complex is combined the immune complex that forms similar double-antibody sandwich with the monoclonal antibody that detects the band place, remaining fluorescent-labeled antibody moves to the control line reaction and forms band, positive result;
(2) detect by fluorescence detection device;
(3) result's judgement: detect band and do not show that quality control band shows band, shows that test strips is effective, testing sample does not contain the antigen of surveying; Detect band and show band, quality control band shows band, shows that testing sample contains to survey to some extent antigen; Detect band and do not show that quality control band does not show, shows that test strips is invalid; Detect band and show band, quality control band does not show, shows that test strips is invalid.
The present invention combines the signal amplifying system technology of glucosan-antibody-fluorescein mixed mark with traditional immunochromatography technique, develop a kind ofly to have highly sensitive fluorescent quantitation test paper and be used for the diagnosing acute miocardial infarction.This fluorescent quantitation test paper changes the antigenic content that comes in the response sample by fluorescence intensity, can realize the special quantitative detection of antigen.The Severity of this early diagnosis for miocardial infarction, miocardial infarction, therapeutic scheme are selected and the prognosis judgement has great importance.The signal amplifying system technology of the glucosan-antibody in this fluorescent test paper-fluorescein mixed mark can be increased to 0.1ng/ml with sensitivity simultaneously, and micro substance is epochmaking in serum for detecting for this.In addition, this high sensitivity fluorescent quantitation diagnose test paper got final product observations in 3-5 minute, the sample consumption is few, can detect human muscle hemoglobin in the human serum/blood plasma, human troponin I and three indexs of people's creatine kinase isozyme, and this is epochmaking for the early diagnosis miocardial infarction.Product of the present invention provides responsive, special, new tool efficiently for the clinical diagnosis miocardial infarction.
The present invention has following advantage and effect with respect to prior art:
(1) specificity utilizes antigen and antibody idiosyncrasy principle to detect human muscle hemoglobin, Troponin I and creatine kinase isozyme, and its relative specificity can reach respectively 96.6%, 97.0%, 98.0%;
(2) susceptibility, adopt glucosan-antibody-fluorescein mixed mark technology signal amplification technique, the sensitive range of diagnostic reagent can be advanced to 0.1ng/mL from 10-100ng/mL, be that susceptibility has improved 100-1000 doubly, and can realize that micro-myoglobins, Troponin I and creatine kinase isozyme quantitatively detect in the infraction serum;
(3) rapidity can obtain the result in 3-5 minute behind the development point sample of the present invention;
(4) simplicity, development of the present invention is easy and simple to handle, does not need the professional to operate.
Description of drawings
In order to be illustrated more clearly in the embodiment of the invention or technical scheme of the prior art, the below will do the figure of required use in embodiment or the description of the Prior Art to introduce simply, apparently, figure in the following describes only is some embodiments of the present invention, for those of ordinary skills, under the prerequisite of not paying creative work, can also obtain according to these figure other figure.
Fig. 1 is the as a result interpretation synoptic diagram of test strips provided by the present invention, and wherein: T is for detecting band, and C is quality control band.
Fig. 2 is the structural representation of test strips provided by the invention, and wherein: 1 is the absorption of sample pad, and 2 is pad, and 3 is chromatographic film, and 4 is base plate, and 5 is adsorptive pads.
Embodiment
Below in conjunction with figure of the present invention, technical scheme of the present invention is clearly and completely described, obviously, described embodiment only is the present invention's part embodiment, rather than whole embodiment.Based on the embodiment among the present invention, those of ordinary skills belong to the scope of protection of the invention not making the every other embodiment that obtains under the creative work prerequisite.
Principle of the present invention is that detection method involved in the present invention is double antibody sandwich method, one is coated antibody, one is labelled antibody, antigen in the serum is detected, the conventional antibody mark adopts Horseradish Peroxidase Conjugates, but there is the poor problem of susceptibility in horseradish peroxidase-labeled.The fluorescein labelled antibody technology can greatly improve the susceptibility that detects reagent.With regard to labelled antibody, conventional fluorescein-labelled a small amount of fluorescein molecule that only has, antibody is combined with unit, thereby the reading ratio between its positive signal/background signal is excessively low.The present invention uses glucosan-antibody-fluorescein mixed mark technology especially.Can contain 1000 divinyls μ lfone(divinyl sulfones based on each dextran molecule) reactive group, except divinyls μ lfone reactive group self coupling of small part, form the structure of similar mouth mask, the divinyls μ lfone reactive group of the overwhelming majority can carry out respectively coupling with fluorescein or antibody in each dextran molecule.Therefore, first glucosan activation is combined with hundreds of fluorescein molecule, in conjunction with after glucosan-fluorescein potpourri be combined with antibody again, utilize the hundreds of raising of reading ratio between this technology positive signal/background signal.
Fluorescence immunoassay test paper provided by the present invention, principle comprise double antibody sandwich method and two kinds of immunological methods of indirect method.Double antibody sandwich method is the reaction method that detects band, determined antigen at the pad place antibody in glucosan-antibody-fluorescein potpourri be combined, form immune complex, this immune complex carries out swimming and carries out combination with detection band antibody on the film in chromatographic film, forms the immune complex that solidifies; Indirect method is the reaction method of quality control band, and the antibody of glucosan-fluorescein potpourri mark carries out combination in swimming on the film and with antiantibody on the quality control band, forms the immune complex that solidifies.
Purpose of the present invention is achieved through the following technical solutions: a kind of test paper that detects acute myocardial infarction of the present invention comprises base plate, is attached to the absorption of sample pad, pad, chromatographic film and the adsorptive pads that closely link to each other successively on the base plate; Wherein, pad is coated with glucosan-antibody-fluorescein mixed mark thing; Have one on the chromatographic film and detect band and a quality control band, detection is with and is fixed with monoclonal antibody; Quality control band be fixed with can with the rabbit anti-mouse igg antibody of glucosan-antibody-fluorescein mixed mark thing specific bond.By being fixed with human muscle hemoglobin, human troponin I and the people's creatine kinase isozyme in the monoclonal antibody detection people whole blood on the detection paper band.On each antigen paper bar control line C(Control is arranged all) and detection line T(Test), control line is coated with rabbit anti-mouse igg antibody, one of on the detection line in coated anti-myoglobins, Troponin I and the creatine kinase isozyme monoclonal antibody, form glucosan-antibody-fluorescein mixed mark thing-antigen-antibody complex, remaining fluorescent-labeled antibody moves to control line (C line) reaction and forms band, positive result.If band does not appear in the respective item detection zone, then the respective item result is negative, and control line C place forms band, shows the detection system normal operation.
Monoclonal antibody of the present invention, antibody are one of anti-myoglobins, Troponin I and creatine kinase isozyme.
The preparation of wherein said monoclonal antibody:
Monoclonal antibody of the present invention is anti-myoglobins, Troponin I and creatine kinase isozyme monoclonal antibody, and they make through Fusion of Cells, screening and cloning cultivation more respectively with above-mentioned three kinds of antigen immune mouse.
Preparation process is:
(1) uses monoclonal antibody immunity antigen immune mouse; With after monoclonal antibody antigen and the emulsification of equivalent Freund's complete adjuvant, adopt subcutaneous multi-point injection (100 μ g/0.2mL/ only) mode that the female BALB/c mouse in 44 ages in week is carried out subcutaneous inoculation respectively; Just exempt from rear booster immunization for several times.
(2) screen anti-monoclonal antibody;
A, with 1X10
8Splenocyte and 2X10
7Myeloma cell SP2/0 is mixed in the 50mL fusion pipe, adds the DMEM nutrient culture media to 30mL.The centrifugal 7min of 1000rpm is with as far as possible exhaustion of supernatant;
B, touch fusion pipe bottom at palm, make sedimentation cell loose evenly, put preheating in 40 ℃ of water-baths.With the 1mL suction pipe in 1 minute, add be preheated to 40 ℃ 50%, the limit edged shakes mixing gently, adds the incomplete DMEM nutrient culture media that 25mL is preheated to 37 ℃ with the 5mL suction pipe in 90 seconds, room temperature leaves standstill 10min, centrifugal 7 minutes of 800rpm abandons supernatant;
C, adding 5Ml HAT complete medium, the pressure-vaccum sedimentation cell suspends and mixing it gently, then adds the HAT nutrient culture media to 90mL.Divide to be filled to and merge the 96 porocyte culture plates that contain feeder cells of cultivating the previous day, every hole 100 μ L; Culture plate is put in 37 ℃ of 5%CO2 incubators and is cultivated;
D, be cultured to the 5th day, with the HT complete medium 1/2HAT nutrient solution in the hole that swaps out.Be cultured to the 7th day, with HT nutrient culture media all nutrient solutions that swap out in the hole;
E, every day are observed the Growth of Hybridoma Cell situation, and when treating that hybridoma grows to 10% above hole floorage, the sucking-off nutrient solution carries out antibody test; Suppress the ELISA method with competition, filter out the hybridoma of secretion antibacterium lipopolysaccharides and fat-soluble protein monoclonal antibody.
(3) ascites production, the selection standard BALB/c mouse is carried out the mouse peritoneal injection with norphytane first, and every mouse is according to 5X10 after the week
6Hybridoma is inoculated in the mouse peritoneal and goes.Gather ascites after 1 week, place 37 ℃ to leave standstill 2 hours ascites after, the centrifugal 10min of 13000rpm removes cell component and other sediment, collects supernatant, after glass fiber pellets, collects the ascites of clarification.
(4) antibody purification adopts caprylic acid-ammonium to carry out preliminary purification, get the 1mL dialysis after crude extract add the 1mL prepacked column, wash with the PBS of pH=7.4, the 100mM glycocoll of the 1mL of adding pH=2.7 carries out wash-out, repeats 6 times.Collect efflux, the 1mL/ pipe, and with 50 μ l 1M Tris neutralization reactions.Adopt SDS-PAGE its purity of electrophoresis detection and concentration, use simultaneously ELISA to detect antibody purification and tire.
The preparation of wherein said rabbit anti-mouse igg antibody:
Preparation process is:
(4) with the immunogene that contains mouse IgG immunizing rabbit repeatedly;
(5) collect, separate the serum that obtains containing rabbit anti-mouse igg antibody;
(6) antibody serum is joined purifying obtains rabbit anti-mouse igg antibody in the affinity column.
Wherein said glucosan-antibody-fluorescein mixed mark thing preparation
Preparation process is:
(1) with fluorescein molecule and the reaction of the dextran molecule take hydrophilic chain as skeleton, obtains the dextran molecule of combined with fluorescent element;
(2) in connection with dextran molecule and the antibody response of fluorescein, purifying obtains glucosan, antibody and fluorescein mixed mark thing;
Described antibody is a kind of in anti-myoglobins, Troponin I and the creatine kinase isozyme monoclonal antibody, determines to use the monoclonal antibody of corresponding antigens according to the antigen that detects.
Described glucosan is preferably molecular weight 20,000～30,000 glucosan;
Described fluorescein is preferably fluorescein isothiocynate;
Described purifying preferably carries out separation and purification by gel chromatography; Because there is significant difference in molecular weight between glucosan-antibody-fluorescein mixed mark thing and other materials (glucosan, antibody and fluorescein), thereby can carry out chromatography with the molecular sieve purification material, glucosan-antibody-fluorescein mixed mark thing is because molecular weight is large, at first from purification column wash-out out, then just slowly wash-out is out from purification column after above-mentioned peak value for the glucosan that relative molecular weight is less, antibody and fluorescein;
Described glucosan-antibody-fluorescein mixed mark thing is by following preferred preparation process:
(1) preparation of the dextran molecule of combined with fluorescent element: use the organic solvent dissolution fluorescein, under the magnetic agitation condition, dropping is in containing the acetum of glucosan, and the lucifuge reaction is so that the carbon atom on the fluorescein and the reaction of the divinyl sulfone on the glucosan are in order to carry out the mark coupling; Reaction transfers to 8.8-9.2 with the pH value after finishing, and is centrifugal, gets precipitation, obtains the dextran molecule of combined with fluorescent element; Wherein, fluorescein and glucosan are that the mass ratio of 8-10:1 carries out proportioning by scope.
(2) preparation of glucosan-antibody-fluorescein mixed mark thing head product: the precipitation that washing step (1) obtains, use again the acetum dissolution precipitation, then the pH value is adjusted to 4.8-5.2; Under the magnetic agitation condition, be added dropwise to antibody-solutions, the amount that antibody adds is pressed the 1mg fluorescein and is added the calculating of 100mg immunoglobulin (Ig), obtains glucosan, antibody and fluorescein mixed mark thing head product after reacting.
(3) preparation of glucosan-antibody-fluorescein mixed mark thing: use sephadex dress post, with pH scope 6.8-7.2,0.002-0.01M phosphate buffer (PB) balance; With glucosan, antibody and the fluorescein mixed mark thing head product loading that step (2) obtains, use again pH6.8-7.2,0.002-0.01M phosphate buffer wash-out, when yellow-green fluorescence appears at eluent, collect whole yellow-green fluorescence solution; With the dialysis of yellow-green fluorescence solution, obtain glucosan-antibody-fluorescein mixed mark thing;
Organic solvent described in the step (1) is preferably absolute ethyl alcohol;
Fluorescein described in the step (1) is preferably fluorescein isothiocynate;
The concentration that fluorescein described in the step (1) is dissolved in organic solvent is preferably 1-2mg/ml;
Glucosan described in the step (1) is preferably molecular weight 20,000～30,000 glucosan;
The concentration of glucosan is preferably 5-10mg/ml in the acetum that contains glucosan described in the step (1); The concentration of acetic acid is preferably 0.2-0.5mol/L;
The time of the reaction described in the step (1) is preferably 2-6 hour;
PH described in the step (1) preferably regulates by NaOH; More preferably regulate by 10mol/L NaOH solution;
Centrifugal condition described in the step (1) is preferably 8000-10000rpm5-10 minute;
Washing described in the step (2) preferably uses distilled water to wash, wash to liquid be colourless till;
The concentration of the acetum described in the step (2) is preferably mass percent 1-5%;
The concentration 1-2mg/ml that the consumption of the acetum described in the step (2) is preferably by the dissolving fluorescein calculates consumption.
PH described in the step (2) preferably regulates by NaOH; More preferably regulate by 10mol/L NaOH solution;
The concentration of the antibody-solutions described in the step (2) is preferably 15-25mg/mL;
The time of the reaction described in the step (2) is preferably 15-25 minute;
Sephadex described in the step (3) is preferably the Sephadex-G50 gel;
The speed of the wash-out described in the step (3) is preferably 1ml/min;
Bag filter, 0.01mol/L phosphate buffer (pH7.9-8.1), (0～4 ℃) that the condition of the dialysis described in the step (3) is preferably molecular cut off 10,000 spend the night.
The preparation specific embodiment of described glucosan-antibody-fluorescein mixed mark thing:
(1) preparation of the dextran molecule of combined with fluorescent element: take by weighing 10mg fluorescein isothiocynate (FITC), be dissolved in the 10mL absolute ethyl alcohol, dropwise joining 20mL glucosan concentration under the magnetic agitation condition is in the acetum (0.2mol/L) of 40mg/ml, lucifuge reaction 4 hours is reacted in order to carry out the mark coupling carbon atom and the divinyl sulfone on the glucosan on the FITC.NaOH with 10mol/L regulates pH value to 9, and centrifugal 9000rpm, gets precipitation at 7 minutes time.
(2) preparation of glucosan-antibody-fluorescein mixed mark thing head product: use the distilled water washing precipitation, until filtrate clarification be colourless till.To precipitate again with the dissolving of mass percent 2% acetum, regulate pH value to 5 with 10mol/L NaOH, dropwise add 10mL antibody protein solution under the magnetic agitation condition, concentration is 20mg/mL, reacts to obtain glucosan, antibody and fluorescein mixed mark thing head product after 20 minutes;
(3) preparation of glucosan-antibody-fluorescein mixed mark thing: with Sephadex-G50 dress 25cm chromatographic column, the about 18cm of gel precipitation from mouth of pipe 7cm, avoids gel column to kill, with about 10 minutes of the 0.005M phosphate buffer balance wash-out of pH=7.0, flow control was 1ml/ minute.Draw gel column on the above-mentioned marking fluid, attention can not make cylinder kill.With the 0.005M phosphate buffer wash-out of pH=7.0, flow control is 1ml/ minute.When yellow-green fluorescence appears at eluent, collect whole yellow-green fluorescence solution with test tube.The yellow fluorescence eluant solution is slower, with about 15 minutes of distilled water wash-out, until yellow fluorescence disappears, stop wash-out after, the post inner gel is recovered in the gel bottle.Above-mentioned glucosan-antibody-fluorescein mixed mark thing is placed in the PBS damping fluid of 0.01M of pH=7.4 through dialysis, adds 0.1% NaN3,1%BSA, and 4 ℃ keep in Dark Place.
Wherein pad preparation
Preparation process:
(1) glucosan-antibody-fluorescein mixed mark thing 20mg is diluted in the PBS damping fluid of pH7.40.01M of 2000ml;
(2) damping fluid adds 0.1% NaN3 and 1%BSA, prepares the concentration 0.01mg/ml of above-mentioned mixed mark thing;
(3) the antibody pad is soaked in the mixed mark thing of 2000ml, is controlled at 2-5 minute action time, take out rear 37 ℃ of air-casing formula oven for drying, it is for subsequent use to keep in Dark Place under 45% humidity.
The preparation of test paper
Preparation process:
1, a kind of test strips material requested of production is as follows:
1. nitrocellulose membrane: by U.S. Millipore/NC import, the specification of nitrocellulose membrane is 30cmx3m/HAHY00010:
Article one, the required cellulose nitrate membrane area of gold label test strip is: 2.5cm * 0.9cm.
Producing the required nitrocellulose membrane total area of 100,000 person-portion high sensitivity fluorescent quantitation test strips is:
2.5cm * 0.9cm * 100,000=2.25 * 105cm
2
Produce the Total Volumes of the required nitrocellulose membrane of 100,000 person-portion test strips:
2.25 * 105cm
2/ 30 * 300cm
2=25 volumes
2. detect required antibody on the T line:
The total amount of antibody: 0.5mg/ml * 2.0ul/cm * 0.3cm * 100,000=30mg
3. detect rabbit anti-mouse igg on the control line C line:
The total amount of rabbit anti-mouse igg: 1mg/ml * 2.0ul/cm * 0.3cm * 100,000=60mg
4. be used for the glucosan-monoclonal antibody of label pad-fluorescein mixed mark thing:
The total amount of required monoclonal antibody: 0.01mg/ml * 20ul * 100,000=20mg
Described antibody is a kind of in anti-myoglobins, Troponin I and the creatine kinase isozyme monoclonal antibody.
2. the spray of the control line on the tunica fibrosa, detection line
With the above-mentioned anti-myoglobins of nitrocellulose membrane difference spray 60ml, Troponin I and the creatine kinase isozyme monoclonal antibody for preparing, concentration is 0.5mg/ml, and 2.0 μ l/cm spray in the position of nitrocellulose membrane T line.Then simultaneously with the 60ml sheep anti-mouse igg, concentration is 1mg/ml, and 2.0 μ l/cm spray in the position of nitrocellulose membrane C line.
3, label pad, the assembling of sample pad
With the above-mentioned glucosan-antibody for preparing-fluorescein-labelled potpourri 20mg, preparation concentration is 0.01mg/ml immersion treatment pad material, obscure for avoiding producing, hereby explain: above-mentioned 20mg and concentration 0.01mg/ml refer to total amount and the concentration of antibody in the mixed mark thing.Rather than fluorescein, the total amount of glucosan and concentration.Then be assembled into the position of pad on the above-mentioned nitrocellulose membrane after soaked label being dried, nitrocellulose membrane sprayed and the C line was arranged, the T line this moment.At last, same principle is assembled into sample pad the position of sample pad in the test strips.
4, cut and finished product assembling
Absorption of sample pad, pad, the nitrocellulose membrane that is coated with detection line and control line, adsorptive pads, head and the tail are connected mutually, optimize convergence condition, are fixed in order on the adhesive sticker base plate; Cut into inch strips the assembling test strips by certain specification with cutting machine.
To carry out the finished product calibrating for test paper, main specificity, susceptibility and stability test to test paper
1, the specificity of fluorescence immunoassay quantitative test paper check
Product contrast of the present invention detects the material sample that may exist in positive sample (containing myoglobins, creatine kinase isozyme and Troponin I) and other clinical blood samples, to determine the specificity of this product.These materials comprise: human serum albumin (100mg/ml), tire red pigment (5mg/ml), haemoglobin (10mg/ml), cholesterol (5mg/ml), triglyceride (15mg/ml), human skeletal's Troponin I (10.000ng/ml), cardiac troponin (20,000ng/ml).Test sample is added drop-wise on the immunofluorescence test paper of the present invention, utilizes portable fluorescence detector (ESE-Quant FLUO, QIAGEN company product) to detect, and test findings shows that the positive sample reaction is positive.Other samples are without being negative, illustrate above-mentioned substance shown in do not have cross reaction with product of the present invention under the concentration.
2, product sensitivity Detection test
Aforementioned positive sample is pressed the variable concentrations preparation, and carry out fluorescent test paper and detect.Testing result shows: the sensitivity of three kinds of myoglobins, creatine kinase isozyme and the detections of Troponin I index all reaches the level of 0.1ng/ml.Testing result is as shown in the following chart:
Myoglobins immunofluorescence detection paper result
Troponin I immunofluorescence detection paper result
Creatine kinase isozyme immunofluorescence detection paper result
3. stability test
Sealing is stored in 4 ℃ of test paper detected once every 7 days, carry out stability test, the result shows 120 days and still can detect the positive.
The above; be the specific embodiment of the present invention only, but protection scope of the present invention is not limited to this, anyly is familiar with those skilled in the art in the technical scope that the present invention discloses; can expect easily changing or replacing, all should be encompassed within protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion by described protection domain with claim.
Claims (18)
1. a test paper that detects acute myocardial infarction is characterized in that: comprise base plate, be attached to the absorption of sample pad, pad, chromatographic film and the adsorptive pads that closely link to each other successively on the base plate; Wherein, pad is coated with glucosan-antibody-fluorescein mixed mark thing; Have one on the chromatographic film and detect band and a quality control band, detection is with and is fixed with monoclonal antibody; Quality control band be fixed with can with the rabbit anti-mouse igg antibody of glucosan-antibody-fluorescein mixed mark thing specific bond.
2. a kind of test paper that detects acute myocardial infarction according to claim 1, it is characterized in that: described monoclonal antibody is one of anti-myoglobins, Troponin I and creatine kinase isozyme.
3. a kind of test paper that detects acute myocardial infarction according to claim 1, it is characterized in that: the preparation process of wherein said monoclonal antibody is:
(1) uses monoclonal antibody immunity antigen immune mouse; With after monoclonal antibody antigen and the emulsification of equivalent Freund's complete adjuvant, adopt subcutaneous multi-point injection mode that the female BALB/c mouse in 44 ages in week is carried out subcutaneous inoculation respectively; Just exempt from rear booster immunization for several times;
(2) screen anti-monoclonal antibody;
A, with 1X10
8Splenocyte and 2X10
7Myeloma cell SP2/0 is mixed in the 50mL fusion pipe, adds the DMEM nutrient culture media to 30mL; The centrifugal 7min of 1000rpm is with as far as possible exhaustion of supernatant;
B, touch fusion pipe bottom at palm, make sedimentation cell loose evenly, put preheating in 40 ℃ of water-baths, with the 1mL suction pipe in 1 minute, add be preheated to 40 ℃ 50%, the limit edged shakes mixing gently, adds the incomplete DMEM nutrient culture media that 25mL is preheated to 37 ℃ with the 5mL suction pipe in 90 seconds, and room temperature leaves standstill 10min, centrifugal 7 minutes of 800rpm abandons supernatant;
C, adding 5Ml HAT complete medium, the pressure-vaccum sedimentation cell suspends and mixing it gently, then adds the HAT nutrient culture media to 90mL, divides to be filled to the 96 porocyte culture plates that contain feeder cells that merge cultivation the previous day, every hole 100 μ L; Culture plate is put in 37 ℃ of 5%CO2 incubators and is cultivated;
D, be cultured to the 5th day, with the HT complete medium 1/2HAT nutrient solution in the hole that swaps out, be cultured to the 7th day, with HT nutrient culture media all nutrient solutions that swap out in the hole;
E, every day are observed the Growth of Hybridoma Cell situation, and when treating that hybridoma grows to 10% above hole floorage, the sucking-off nutrient solution carries out antibody test; Suppress the ELISA method with competition, filter out the hybridoma of secretion antibacterium lipopolysaccharides and fat-soluble protein monoclonal antibody;
(3) ascites production, the selection standard BALB/c mouse is carried out the mouse peritoneal injection with norphytane first, and every mouse is according to 5X10 after the week
6Hybridoma is inoculated in the mouse peritoneal and goes; Gather ascites after 1 week, place 37 ℃ to leave standstill 2 hours ascites after, the centrifugal 10min of 13000rpm removes cell component and other sediment, collects supernatant, after glass fiber pellets, collects the ascites of clarification;
(4) antibody purification adopts caprylic acid-ammonium to carry out preliminary purification, get the 1mL dialysis after crude extract add the 1mL prepacked column, wash with the PBS of pH=7.4, the 100mM glycocoll of the 1mL of adding pH=2.7 carries out wash-out, repeats 6 times; Collect efflux, the 1mL/ pipe, and with 50 μ l 1M Tris neutralization reactions.
4. a kind of test paper that detects acute myocardial infarction according to claim 1, it is characterized in that: the preparation process of described rabbit anti-mouse igg antibody is:
(1) with the immunogene that contains mouse IgG immunizing rabbit repeatedly;
(2) collect, separate the serum that obtains containing rabbit anti-mouse igg antibody;
(3) antibody serum is joined purifying obtains rabbit anti-mouse igg antibody in the affinity column.
5. a kind of test paper that detects acute myocardial infarction according to claim 1, it is characterized in that: described glucosan-antibody-fluorescein mixed mark thing preparation process is:
(1) with fluorescein molecule and the reaction of the dextran molecule take hydrophilic chain as skeleton, obtains the dextran molecule of combined with fluorescent element;
(2) in connection with dextran molecule and the antibody response of fluorescein, purifying obtains glucosan, antibody and fluorescein mixed mark thing.
6. a kind of test paper that detects acute myocardial infarction according to claim 5, it is characterized in that: described glucosan is preferably molecular weight 20,000～30,000 glucosan.
7. a kind of test paper that detects acute myocardial infarction according to claim 5, it is characterized in that: described fluorescein is preferably fluorescein isothiocynate.
8. a kind of test paper that detects acute myocardial infarction according to claim 5, it is characterized in that: described purifying preferably carries out separation and purification by gel chromatography.
9. a kind of test paper that detects acute myocardial infarction according to claim 1 is characterized in that: described glucosan-antibody-fluorescein mixed mark thing preparation process:
(1) preparation of the dextran molecule of combined with fluorescent element: use the organic solvent dissolution fluorescein, under the magnetic agitation condition, dropping is in containing the acetum of glucosan, and the lucifuge reaction is so that the carbon atom on the fluorescein and the reaction of the divinyl sulfone on the glucosan are in order to carry out the mark coupling; Reaction transfers to 8.8-9.2 with the pH value after finishing, and is centrifugal, gets precipitation, obtains the dextran molecule of combined with fluorescent element;
(2) preparation of glucosan-antibody-fluorescein mixed mark thing head product: the precipitation that washing step (1) obtains, use again the acetum dissolution precipitation, then the pH value is adjusted to 4.8-5.2; Under the magnetic agitation condition, be added dropwise to antibody-solutions, the amount that antibody adds is pressed the 1mg fluorescein and is added the calculating of 100mg immunoglobulin (Ig), obtains glucosan, antibody and fluorescein mixed mark thing head product after reacting;
(3) preparation of glucosan-antibody-fluorescein mixed mark thing: use sephadex dress post, with pH scope 6.8-7.2,0.002-0.01M phosphate buffer balance; With glucosan, antibody and the fluorescein mixed mark thing head product loading that step (2) obtains, use again pH 6.8-7.2,0.002-0.01M phosphate buffer wash-out, when yellow-green fluorescence appears at eluent, collect whole yellow-green fluorescence solution; With the dialysis of yellow-green fluorescence solution, obtain glucosan-antibody-fluorescein mixed mark thing.
10. a kind of test paper that detects acute myocardial infarction according to claim 10 is characterized in that: described element and glucosan are that the mass ratio of 8-10:1 carries out proportioning by scope.
11. a kind of test paper that detects acute myocardial infarction according to claim 10 is characterized in that: the organic solvent described in the step (1) is preferably absolute ethyl alcohol, and fluorescein is preferably fluorescein isothiocynate.
12. a kind of test paper that detects acute myocardial infarction according to claim 10 is characterized in that: in the step (1), the concentration that described fluorescein is dissolved in organic solvent is preferably 1-2mg/ml; Described glucosan is preferably molecular weight 20,000～30,000 glucosan; The concentration of glucosan is preferably 5-10mg/ml in the described acetum that contains glucosan; The concentration of acetic acid is preferably 0.2-0.5mol/L; The time of described reaction is preferably 2-6 hour; Described pH regulates by NaOH, and centrifugal condition is preferably 8000-10000rpm 5-10 minute.
13. a kind of test paper that detects acute myocardial infarction according to claim 13 is characterized in that: described pH regulates by 10mol/L NaOH solution.
14. a kind of test paper that detects acute myocardial infarction according to claim 10 is characterized in that: in the step (2), described washing preferably uses distilled water to wash, wash to liquid be colourless till; The concentration of described acetum is preferably mass percent 1-5%; The concentration 1-2mg/ml that the consumption of described acetum is preferably by the dissolving fluorescein calculates consumption, and described pH regulates by NaOH; The concentration of described antibody-solutions is preferably 15-25mg/mL; The time of described reaction is preferably 15-25 minute.
15. a kind of test paper that detects acute myocardial infarction according to claim 10 is characterized in that: in the step (3), described sephadex is preferably the Sephadex-G50 gel; The speed of described wash-out is preferably 1ml/min; Bag filter, 0.01mol/L phosphate buffer (pH7.9-8.1), (0～4 ℃) that the condition of described dialysis is preferably molecular cut off 10,000 spend the night.
16. a kind of test paper that detects acute myocardial infarction according to claim 1 is characterized in that: pad preparation process wherein:
(1) glucosan-antibody-fluorescein mixed mark thing 20mg is diluted in the PBS damping fluid of pH7.40.01M of 2000ml;
(2) damping fluid adds 0.1% NaN3 and 1% BSA, prepares the concentration 0.01mg/ml of above-mentioned mixed mark thing;
(3) the antibody pad is soaked in the mixed mark thing of 2000ml, is controlled at 2-5 minute action time, take out rear 37 ℃ of air-casing formula oven for drying, it is for subsequent use to keep in Dark Place under 45% humidity.
17. a kind of method for preparing test paper that detects acute myocardial infarction according to claim 1 is characterized in that: comprise following steps:
(1) with dilution glucosan-antibody-fluorescein mixed mark thing is diluted to concentration to the 0.01-0.03mg/ml scope, then is immersed in the dilution in connection with pad, after oven dry or vacuum freeze drying, refill and be fitted on the test strips base plate; Consisting of of dilution: add mass percent 0.1% NaN3, mass percent 1% BSA in the PBS damping fluid of pH7.4 0.01M;
(2) in chromatographic film two bands are set, one is detected band and a quality control band; Immobilized monoclonal antibody is with in detection, and described clonal antibody is a kind of of anti-myoglobins, Troponin I and creatine kinase isozyme monoclonal antibody; Quality control band is rabbit anti-mouse igg antibody fixedly;
(3) stick absorption of sample pad and pad in a side of the chromatographic film of step (2), opposite side sticks adsorptive pads; The detection band of absorption of sample pad, pad, chromatographic film, quality control band and the adsorptive pads of chromatographic film are arranged successively.
18. a kind of test paper application process that detects acute myocardial infarction according to claim 1 is characterized in that: comprise following steps:
(1) adds the test serum sample at the absorption of sample pad; Under capillary action, sample liquid is to adsorptive pads one end swimming, form immune complex at pad place and glucosan-antibody-fluorescein mixed mark thing, again further swimming, immune complex is combined the immune complex that forms similar double-antibody sandwich with the monoclonal antibody that detects the band place, remaining fluorescent-labeled antibody moves to the control line reaction and forms band, positive result;
(2) detect by fluorescence detection device;
(3) result's judgement: detect band and do not show that quality control band shows band, shows that test strips is effective, testing sample does not contain the antigen of surveying; Detect band and show band, quality control band shows band, shows that testing sample contains to survey to some extent antigen; Detect band and do not show that quality control band does not show, shows that test strips is invalid; Detect band and show band, quality control band does not show, shows that test strips is invalid.
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| CN107907514A (en) * | 2017-11-01 | 2018-04-13 | 上海凯创生物技术有限公司 | CTnI/myoglobins/creatine kinase isozyme near-infrared fluorescent detection reagent card, kit and application thereof |
| CN108303551A (en) * | 2018-02-07 | 2018-07-20 | 浙江聚康生物工程有限公司 | Human cardiac troponin I/three-in-one assay kit of myoglobins/creatine kinase isozyme and preparation method |
| CN110520736A (en) * | 2019-07-15 | 2019-11-29 | 中国科学院苏州生物医学工程技术研究所 | A kind of functionalization biology multilayer hole film immune carrier, preparation method, application |
| WO2021007738A1 (en) * | 2019-07-15 | 2021-01-21 | 中国科学院苏州生物医学工程技术研究所 | Functionalized biological multilayer porous membrane immune carrier, preparation method, and application |
| CN110520736B (en) * | 2019-07-15 | 2022-07-22 | 中国科学院苏州生物医学工程技术研究所 | Functionalized biological multilayer porous membrane immune carrier, preparation method and application |
| CN110646616A (en) * | 2019-09-05 | 2020-01-03 | 桂林理工大学 | Hypersensitivity fluorescence quenching immunosensor for detecting human cTnI in serum and detection method |
| CN111089958A (en) * | 2019-12-26 | 2020-05-01 | 江苏美克医学技术有限公司 | P16 based on glucan signal amplificationINK4aChemiluminescence kit |
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