CN101762699A - Magnetic immuno-chromatographic test paper strip for quantitatively detecting tumor associated antigen 125 in blood and preparation method thereof - Google Patents
Magnetic immuno-chromatographic test paper strip for quantitatively detecting tumor associated antigen 125 in blood and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a magnetic immuno-chromatographic test paper strip for quantitatively detecting tumor associated antigen 125 in blood and a preparation method thereof. A coating film, a magnet particle pad combined with tumor associated antigen 125 antibodies, a sample pad and a water absorbing pad are sequentially and alternately stuck on a base plate in a staggering way at intervals of 2mm, and then the upper layer is covered by a transparent plastic sealing film to form a test paper strip. A tumor associated antigen 125 antibody detection line and a quality control line are pre-coated on the coating film. The invention introduces the magnetic immuno-chromatographic technology and the fluorescein isothiocyanate system into the quantitative detection of tumor associated antigen 125, and has the advantages that the detection sensitivity is greatly improved, the accurate quantitative results can be given, the operation is simple and convenient and the diagnosis is rapid.
Description
Technical field
The invention belongs to field of medical examination, particularly relate to the magnetic immuno-chromatographic test paper strip and the preparation method of tumor-associated antigen 125 in a kind of detection by quantitative blood.
Background technology
Tumor-associated antigen 125 (CA125), molecular weight can reach between 220000～1000000, and glycosyl content only accounts for 24% of total protein quality.CA125 is prepared from by mouse-anti people palilate capsule gamogenetic egg nest epithelial cell line OC125.It is that tumour exists and the reliability index that recurs that change of serum C A125 level raises.CA125 can obviously raise in 80～90% the epithelial ovarian cancer, carcinoma of endometrium, clear cell carcinoma, carcinoma of fallopian tube and do not break up that CA125 content also can obviously raise in the ovarian cancer patients; Mucous ovarian cancer patient's positive rate is then lower, and CA125 is valuable especially in the good pernicious discriminating of ovary enclosed mass, and susceptibility is 78%, specificity 95%, positive prediction are worth 82%, negative predictive value 91%.CA125 and CEA simultaneous determination calculate both ratio, can improve susceptibility and specificity that oophoroma detects.The malignant tumour of non-oophoroma class can be respectively up to 73%, 70%, 53%, 27%, 22% and 20% as the positive rate of CA-125 among pancreas, liver, stomach, intestines, uterus and the mammary gland cancer patient.Yet slight the rising is also shown in 1% healthy women the CA-125 serum-concentration, 3～6% optimum ovary illness or non-tumor patient, comprise initial 3 months of pregnancy period, menstrual period, mullerianosis, fibrosis of uterus, fibroid, optimum ovarioncus, acute salpinitis, acute pancreatitis, hepatopathy, pleuroperitoneum and pericardosis etc., should note getting rid of.Diagnose the susceptibility of recurrence to be lower than specificity but it is to be noted raises with change of serum C A125 level, i.e. the change of serum C A125 prompting recurrence to a great extent that raises does not show no tumour progression but normal CA125 neither shows no tumour existence yet.
The diagnostic techniques of CA125 mainly comprises in the present blood: radiommunoassay (RIA), enzyme-linked immuno assay (ELISA), chemiluminescence immune assay (CLIA), time resolved fluoro-immunoassay, methods such as colloidal gold immunochromatographimethod, but these methods all have separately characteristics and deficiency: radio-immunity is clinical method relatively more commonly used, advantage is that the result is more accurate, range of linearity broad, shortcoming is a complex operation, length consuming time, and radioactively labelled substance can produce harm to the operator, and can produce environmental pollution, be substituted by additive method gradually at present.ELISA and chemiluminescence and time-resolved fluorescence method principle are similar, it is variant aspect sensitivity for no other reason than that final detection system is distinguished to some extent, robotization, in enormous quantities, detection by quantitative have been realized at present, but exist between method result difference big, shortcoming such as the range of linearity is narrower, simultaneously, robotization detects and is monopolized by external large manufacturer at present, instrument and equipment costliness, and be not suitable for single part and less batch detection usefulness has limited their application in basic hospital, clinic greatly.Occur colloidal gold immunity chromatography in recent years and detected CA125, fast, convenient, single part of operation, but shortcoming is to realize sxemiquantitative, can only indicate a probable ranges, can't realize accurate quantification, monstrosity need use additive method to check detection, has increased testee's the cost of seeking medical advice, big difficulty is arranged aspect marketing, be difficult to be extensive use of.
(Mgnetic ImmunoChromatographic Test MICT) is a kind of single part of fast quantification detection technique that occurs in recent years to magnetic immuno-chromatographic.Be to replace traditional label (collaurum, latex particle etc.) to carry out immunochromatography, be combined in biochemical substances on the super-paramagnetism nano particulate by detection detection by quantitative data to biological sample are provided with supperparamagnetic particles (superPMPs).Come the amount of presentation markup by detecting the measurement magnetic field intensity, adopt the typical curve of the immune complex-magnetic field intensity of mark, thereby reach quantitative purpose in sample area.This technology is compared with conventional art has following advantages: 1) sensitivity for analysis: than the sensitive 10-100 of all kinds of range estimation quick diagnosis methods doubly; 2) analysis speed: can in 15 seconds, measure the nearly data of 6 site of analysis; 3) linear range: in 4 order of magnitude concentration ranges, be linear; 4) the used magnetic detecting instrument adopts the solid phase element, and miniaturized design is had a style of one's own, independent operating, and volume is little, and is easy and simple to handle; 5) the super-paramagnetism nano particulate can not decayed in time by polymer coating; Independently quick diagnosis chromatogram card (MAR Cassette) can directly insert the MAR detector, for the integration of many quick reagents for clinical diagnosis at present provides development space widely; But also personnel or the contingent cross pollution of instrument in the operating process, the security that has improved analysis and process have been avoided.This technology has been inherited traditional immunochromatographic method (collaurum, latex particle etc.) easy to be quick, the advantage of single part of operation, it is low to have remedied traditional immunochromatography technique sensitivity again, can only be qualitative, shortcoming that can not be quantitative, represented current real-time test (Point of Care Test, POCT) direction of technical development and trend are once appearance, development has become the first-selection that substitutes traditional immunochromatography technique at present rapidly.
Fluorescein isothiocynate (FITC) is introduced in the magnetic immuno-chromatographic, in the sensitivity that has greatly improved detection method, provided a kind of fabulous current techique platform again, in large-scale production, reduced markers step, reduced variable factor.
Summary of the invention
Purpose of the present invention promptly is with in magnetic immuno-chromatographic technology and the system combined CA125 of the being applied in detection by quantitative of fluorescein isothiocynate (FITC) test strips, with the anti-fluorescein antibody covalent coupling on super paramagnetic particle, FITC-CA125 antibody is mixed with it afterwards as detecting moving phase, with the CA125 antibody sandwich of another strain pairing on nitrocellulose filter as catching solid phase, carry out the detection of sample according to routine immunization chromatography ratio juris, detect in conjunction with simple and easy to do magnetism detector, all drawbacks of aforementioned several detection methods when realizing high-sensitivity detection, both can have been avoided, combine the advantage of aforementioned several method again: can single part detect, also can batch detection, and can provide quantitative result immediately, surveying instrument is simple and reliable, easy and simple to handle, convenient and practical.
For reaching above-mentioned purpose, technical scheme of the present invention is as follows: the magnetic immuno-chromatographic test paper strip of CA125 in a kind of detection by quantitative blood, this test strips is to paste coated film, the magnetic mat of particles that combines CA125 antibody, sample pad, adsorptive pads on interlaced successively 2mm ground on the base plate, cover the test strips that the transparent plastic diaphragm seal assembles on the upper strata then, is coated with CA125 antibody detection line T and nature controlling line C on the coated film in advance.
Wherein said sample pad is through sample pad treating fluid pretreated cellulose membrane, and described sample pad treating fluid is the phosphate buffer (PBS) that contains the 0.02M pH7.0-7.6 of the polyvinyl alcohol (PVA) (PVA) of 1%-5% casein (Casein) and 0.1%-1% and 0.01-0.2% polysorbas20 (Tween-20).
The magnetic immuno-chromatographic test paper strip preparation method of CA125 may further comprise the steps in the above-mentioned detection by quantitative blood:
The preparation of A, FITC-antibody: with CA125 antibody 4 ℃ of dialysed overnight of PBS to 0.02M pH7.0-7.6, adjustment concentration is 2mg/ml, FITC is used the dimethyl formamide dissolving, final concentration is 50mmol, add the FITC solution of aequum with 5: 1 molecule ratios in antibody-solutions, room temperature reaction 1 hour is to 0.02M, 4 ℃ of dialysed overnight of the PBS of pH7.0-7.6 ,-20 ℃ of preservations are standby behind the adding equal-volume glycerine.
The preparation of B, magnetic particle: selecting diameter for use is the super paramagnetic particle of 100-300nm, the mode of using carbodiimide (EDC) covalent coupling with the anti-fluorescein antibody mark to the magnetic particle, the FITC-antibody that mark is good is with 1: 2-1: 5 molecule ratio (mol ratio) is mixed with the magnetic particle of coupling anti-fluorescein antibody, guarantees that the magnetic particle of coupling anti-fluorescein antibody is excessive;
C, use quantitative liquid-jet device to be sprayed on the magnetic mat of particles magnetic particle for preparing with the amount of 25 μ l/cm-50 μ l/cm;
The preparation of D, coated film: use bag to be cushioned liquid and respectively anti-CA 125 coated antibody and FITC-BSA are diluted to 0.5-2mg/ml concentration, use quantitative liquid-jet device respectively with the two with the interval spray printing of 0.5-1.0cm on nitrocellulose filter, dry back immersion in confining liquid and dried 8 hours in the 25-35 degree after 10 minutes, add drying agent and seal up for safekeeping standby;
The processing of E, sample pad: sample pad is put into sample pad Treatment Solution immersion treatment take out 25-35 ℃ of oven dry 8 hours after 1 hour;
The assembling of F, test strips: stick coated film, magnetic mat of particles, sample pad, adsorptive pads successively interlaced 2mm on the transparent plastic base plate, cover the transparent plastic diaphragm seal then on the upper strata and obtain test paper plate, the width cutting promptly obtains test strips as requested.
Further, above-mentioned step B comprises following three steps:
1) preparation of the magnetic particle of coupling anti-fluorescein antibody: use the 50mM that contains 0.1%Tween-20, the PBS washing magnetic particle of pH7.0-7.6, it is 5: 1 (mol ratio) that adding EDC and anti-fluorescein antibody make the molecule ratio of anti-fluorescein antibody and magnetic particle, room temperature reaction 3 hours, add the 0.02M that contains 0.5%BSA, the PBS room temperature sealing of pH7.0-7.6 30 minutes, with above-mentioned PBS washing magnetic particle, use contains 1%PVP, 1%Casein, 0.5%Tween-20, the 50mM of 5% sucrose (pH8.2-9.0) boric acid preserve damping fluid redissolution magnetic particle, and 4 ℃ of preservations are standby;
2) preparation of FITC-CA125 antibody: with CA125 antibody to 0.02M, 4 ℃ of dialysed overnight of the PBS of pH7.0-7.6, adjustment concentration is 2mg/ml, FITC is used dimethyl formamide (DMF) dissolving, and final concentration was 50mM, added the FITC solution of aequum in antibody-solutions with 5: 1 molecule ratios, room temperature reaction 1 hour, to 0.02M, 4 ℃ of dialysed overnight of the PBS of pH7.0-7.6 ,-20 ℃ of preservations are standby behind the adding equal-volume glycerine;
3) the magnetic particle of FITC-antibody and coupling anti-fluorescein antibody mixes, amount with 0.5 μ l/mg magnetic particle adds FITC-CA125 antibody in the magnetic particle solution of coupling anti-fluorescein antibody, fully use behind the mixing and preserve damping fluid with 1: 5-1: 20 ratio (volume ratio) is used mixture diluted fiberglass packing to be sprayed.
Further, among the above-mentioned step C, the spraying method of magnetic particle is: use quantitative spray film device evenly to be sprayed on the glass fibre with the amount of 50 μ l/cm the magnetic particle for preparing, add drying agent after the freeze drying and seal up for safekeeping standby.
Further, among the above-mentioned step D, the preparation method of coated film is: being cushioned liquid (phosphate buffer of 0.02M pH 7.2) with bag is 0.5mg/ml with the CA125 antibody dilution, the FITC-BSA dilution is 1mg/ml, use quantitative spray film device with the amount of 1 μ l/cm with the two with the interval spray printing of 0.6cm on nitrocellulose filter, room temperature is dried after 30 minutes in confining liquid (the 0.02M PBS that contains 0.5%BSA, pH7.0-7.6) immersion in 25-35 ℃ of oven dry 8 hours, added drying agent and seals up for safekeeping standby after 10 minutes in.
Compare with existing quick detection test paper bar, the present invention has the following advantages:
1) by improvement to test strips, first the magnetic immuno-chromatographic technology is introduced in the detection of CA125, in conjunction with magnetism detector, realized the single part of wide region detection by quantitative of CA125, for clinical use provides very big facility.
2) by introducing the fluorescein isothiocynate system, make the preparation process of test strips simplify greatly, be fit to large-scale production.
The present invention is easy and simple to handle, be fit to large-scale production, detect required portable set and also go on the market, therefore can be widely used in short run or single part of unit use of using such as applying unit and some blood sampling scenes, rural area and basic unit clinic in enormous quantities such as hospital, blood station, epidemic prevention station, health check-up.Quantitative Diagnosis for oophoroma has positive meaning.
Description of drawings
Fig. 1 detects the test strips structural representation of CA125 in the blood for magnetic immuno-chromatographic standard measure of the present invention
For further specifying test strips and the preparation method that magnetic immuno-chromatographic standard measure of the present invention detects CA125 in the blood, describe especially exemplified by following embodiment, this embodiment is in order to explain rather than limit by any way the present invention.
Embodiment
The magnetic immuno-chromatographic test paper strip of CA125 as shown in Figure 1 in the detection by quantitative blood of the present invention, this test strips is at base plate 1) on interlaced successively 2mm ground paste to go up coated film 2), combine the magnetic mat of particles 3 of CA125 antibody), sample pad 4), adsorptive pads 5), cover transparent plastic diaphragm seal 6 then on the upper strata) test strips that assembles, coated film 2) on be coated with CA125 antibody detection line T and nature controlling line C in advance.
In specific embodiment, the CA125 that adopts pairing antibody be the monoclonal antibody of monoclonal antibody technique preparation.The principle of utilizing double-antibody sandwich to detect CA125 antigen detects sample; when containing CA125 antigen in the sample to be measured; the antibodies of coupling on antigen meeting elder generation and the magnetic particle; carrying out along with the chromatography effect; bond moves forward and arrives CA125 antibody sandwich line T place; antigen can accumulate in T line place with coated antibody in conjunction with forming the double-antibody sandwich compound once more; in addition; the anti-fluorescein antibody mark magnetic particle in conjunction with FITC-antibody can not continue to move ahead; when arrive accusing line C, thereby FITC-BSA can combine with anti-fluorescein antibody at C line place and occurs the magnetic particle aggregation equally.Entire reaction was carried out in 30 minutes fully, general reaction can be gone up machine-read card after 15 minutes, T line and C line all can produce corresponding magnetic signal value, and the magnetic immuno-chromatographic detector can draw true quantitative results according to the typical curve that actual measured value substitution is default of the 2 D code information on the test card.Whole Card Reader, identification two-dimension code, with the process sequencing fully that measured value substitution preset standard curve draws quantitative values, magnetism detector can directly provide quantitative result.
The preparation method of the magnetic immuno-chromatographic test paper strip of CA125 sees following example in the detection by quantitative blood of the present invention:
The preparation method of the magnetic immuno-chromatographic test paper strip of CA125 and paper box in the detection by quantitative blood
The test strips of present embodiment and the preparation method of paper box may further comprise the steps:
A, Antibody Preparation: select commercial CA125 pairing antibody for use, to 20mM, 4 ℃ of dialysed overnight of PBS of pH7.2 (pH7.0-7.6 all is suitable for) are standby.
The preparation of B, coated film:
Bag is cushioned the preparation of liquid: the phosphate buffer of 0.02M pH 7.2 (PB) is cushioned liquid for bag, and the rearmounted 4 ℃ of preservations of 0.22 μ m filtering with microporous membrane degerming are standby, two weeks of the term of validity.
The preparation of confining liquid: contain the 0.02M of 0.5%BSA, the phosphate buffer (PBS) of pH7.2 (pH7.0-7.6 all is suitable for), it is standby that 0.22 μ m filtering with microporous membrane degerming is placed on 4 ℃ of preservations, one week of the term of validity.
The preparation of coated film: be cushioned liquid (0.02M PB with bag, pH7.2 (pH7.0-7.6 all is suitable for)) be 0.5mg/ml with the CA125 antibody dilution, the FITC-BSA dilution is 1mg/ml, use quantitative spray film device with the amount of 1 μ l/cm with the two with the even spray printing in the interval of 0.6cm on 3.5cm width nitrocellulose filter, room temperature is dried after 30 minutes in confining liquid (the 0.02M PBS that contains 0.5%BSA, pH7.2 (pH7.0-7.6 all is suitable for)) soaks after 10 minutes in and dried 8 hours, add drying agent and seal up for safekeeping standby in the 25-35 degree.
The preparation of C, magnetic particle:
The preparation of PBS damping fluid: with distilled water and sodium dihydrogen phosphate and sodium hydrogen phosphate secure ph is 7.4 (pH7.0-7.6 all is suitable for), concentration is the PBS of 50mM, adding Tween-20 is that 4 ℃ of preservations are standby after 0.1%, the 0.22 μ m filtering with microporous membrane degerming to final concentration, two weeks of the term of validity.
Boric acid is preserved the preparation of damping fluid: use distilled water, boric acid and borax preparation pH are 8.5 (pH8.2-9.0 all is suitable for), and final concentration is the borate buffer of 50mM, add PVP, Casine, Tween-20, sucrose, final concentration is respectively 1%, 1%, 0.5%, 5%, 0.22 4 ℃ of preservations are standby after the degerming of μ m filtering with microporous membrane, one week of the term of validity.
The preparation of the magnetic particle of coupling anti-fluorescein antibody: use the 50mM that contains 0.1%Tween-20, pH7.4 (pH7.0-7.6 all is suitable for) PBS washing magnetic particle, adding EDC and NHS makes the two final concentration be 20mmol, room temperature reaction 1 hour, fully behind the washing magnetic particle, it is 5: 1 (mol ratio) that the adding anti-fluorescein antibody makes the molecule ratio of anti-fluorescein antibody and magnetic particle, room temperature reaction 3 hours, add the 0.02M that contains 0.5%BSA, the 50mM that contains 0.1%Tween-20 is used in the PBS room temperature sealing of pH7.2 (pH7.0-7.6 all is suitable for) 30 minutes, the PBS washing magnetic particle of pH7.4 (pH7.0-7.6 all is suitable for), use contains 1%PVP, 1%Casein, 0.5%Tween-20, the 50mMpH 8.5 of 5% sucrose (pH8.2-9.0 all is suitable for) boric acid is preserved damping fluid, redissolution magnetic particle, 4 ℃ of preservations are standby.
The preparation method of FITC-CA125 antibody is: with CA125 antibody to 0.02M, 4 ℃ of dialysed overnight of PBS of pH7.2 (pH7.0-7.6 all is suitable for), adjustment concentration is 2mg/ml, FITC is used the DMF dissolving, and final concentration was 50mM, added the FITC solution of aequum in antibody-solutions with 5: 1 molecule ratios, room temperature reaction 1 hour, to 0.02M, 4 ℃ of dialysed overnight of PBS of pH7.2 (pH7.0-7.6 all is suitable for) ,-20 ℃ of preservations are standby behind the adding equal-volume glycerine.
Mixing of the magnetic particle of FITC-antibody and coupling anti-fluorescein antibody, amount with 0.5 μ l/mg adds FITC-CA125 antibody in the magnetic particle solution of coupling anti-fluorescein antibody, fully use the preservation damping fluid with 1: 5 ratio mixture diluted fiberglass packing to be sprayed to be used behind the mixing.
The spraying of D, magnetic particle and freeze-drying
That uses BioDot spray film instrument nozzle specially usedly evenly is sprayed at the magnetic particle handled well the amount with 50 μ l/cm on the 0.8cm width fiberglass packing, the frozen overnight drying, add drying agent seal up for safekeeping standby,
The processing of E, sample pad
1.8cm width sample pad is put into sample pad Treatment Solution immersion treatment take out 25-35 ℃ of oven dry 8 hours after 1 hour.
The sample pad treating fluid is to contain the PVA of 1%-5%Casein and 0.1%-1% and the 0.02M of 0.01-0.2%Tween-20, the PBS solution of pH7.2 (pH7.0-7.6 all is suitable for).
The assembling of F, test strips and cutting
Following all operations all must carry out in temperature 20-25 ℃ the room in humidity less than 20%.
The assembling of test paper plate: use BioDot LM5000 type assembling instrument as requested with the 3.5cm coated film, 2.5cm thieving paper, 0.8cm magnetic mat of particles, 1.8cm sample pad are assembled on the 9.8cm width transparent plastic base plate, cover upper strata transparent plastic cover plate, be assembled into test paper plate.
Cutting of test strips: use BioDot CM4000 type cutting cutter that the test paper plate that assembles is cut into the wide finished product test strips of 0.5cm.
The assembling of G, test card
The single part test strips of well cutting of the present invention is placed in the draw-in groove on the plastic bottom card, covers loam cake, use card press machine up and down two plastic clips compress, guarantee that whole test strips is in tensioned state.Adding the drying agent room temperature seals up for safekeeping standby.
H, determine the 2 D code information of this batch
The quantitative magnetic detection card of the name of an article: CA125
Batch: on the assembling date of test card, form is: Year/Month/Day, XXXX/XX/XX
Determining of interpretation standard: get CA125 standard antigen raw material, with the national standard product is that reference standard is demarcated, become 400U/mL with the standard items diluted, 200U/mL, 100U/mL, 50U/mL, 15U/ml, each standard point is done 10 tests, determines the relation of each standard point and Magnetic Measurement value and set up equation to make it to fit to linearity according to statistical method, and this equation and typical curve can make magnetism detector be determined the sample measured value.
Standard items dilution prescription: 20mmol PBS, pH7.2 (pH7.0-7.6 all is suitable for), 0.5%BSA, 0.01%NaN
3
The printing of I, two-dimension code is pasted
With in the above-mentioned 2 D code information input two-dimension code printer and print, two-dimension code is pasted on the ad-hoc location of test card, use two-dimension code paste position detecting device to inspect 2% at random by random samples and guarantee that two-dimension code pastes errorless.
J, finished product packing
The single part test card and that the posts two-dimension code drying prescription of being responsible for a task until it is completed is sealed in the aluminium foil bag, 100 person-portions are that a packing places in the packing box, the a instructions of one box and 1 bottle of 10ml dress chromatography damping fluid are promptly made paper box, and this paper box keeps in Dark Place in room temperature, and the shelf-life is 18 months.
The chromatography buffer formulation is: 1%Tween-20,0.5%Triton X-100,1%NP-40,0.05%NaN
3, 20mmolPBS, pH7.2 (pH7.0-7.6 all is suitable for).
In the step except the preparation of the magnetic particle of coupling anti-fluorescein antibody: it is 1: 2 that anti-fluorescein antibody makes anti-fluorescein antibody and magnetic proportion of particles, and other step is with embodiment 1,
In the step except the preparation of the magnetic particle of coupling anti-fluorescein antibody: it is 1: 10 that anti-fluorescein antibody makes anti-fluorescein antibody and magnetic proportion of particles, and other step is with embodiment 1.
In the blend step except the magnetic particle of FITC-antibody and coupling anti-fluorescein antibody: fully use behind the mixing and preserve damping fluid and with 1: 5 ratio mixture diluted fiberglass packing to be sprayed is used, other step is with embodiment 1.
In the blend step except the magnetic particle of FITC-antibody and coupling anti-fluorescein antibody: fully use behind the mixing and preserve damping fluid and with 1: 20 ratio mixture diluted fiberglass packing to be sprayed is used, other step is with embodiment 1.
The using method of test card of the present invention
1, application of sample
From packing box, take out single part test card, tear the aluminium foil strip packing, test card is placed on the smooth desktop, get 50 μ l sample serum with micropipettor and add in the well on the card, add 50 μ l chromatography damping fluids again, wait question response to carry out 15 minutes.
2, measurement and result output
Test card is inserted the card inserting mouth of magnetic immuno-chromatographic detector, the operation instrument, instrument can read the 2 D code information of card voluntarily, measures and print immediately measurement result, and the yin and yang attribute result can show in print result.
The methodology of embodiment 7 test strips of the present invention is identified
According to manufacturing conventional in this area and vertification regulation the test strips of preparation among the embodiment 1 is examined and determine,
(1) test strips sensitivity experiment
Carry out replication 10 times with the S0 calibration object, its mean value adds that it is the sensitivity of test strips that the twice standard deviation is brought the concentration value of curvilinear equation gained into, and its sensitivity is 2.6U/mL.
(2) test strips specificity experiment
Make cross reaction experiment, cross reacting rate＜0.01% with its analog.
(3) test strips accuracy experiment
Variation within batch
Get basic, normal, high three parts of quality controlled serum samples and carry out 10 hole parallel laboratory tests respectively, calculate the mean value of measured value
And standard deviation (s).By formula
Calculate the coefficient of variation, variation within batch coefficient CV is respectively 6.3%, 4.9%, 4.1%.
Batch variation
Select the blood serum sample of 5 parts of variable concentrations that every part of serum is carried out replication 3 times, calculate its interassay coefficient of variation (CV%), batch variation CV is between 7.21～9.5%.
(4) test strips stability experiment
The test strips preservation condition is 18-26 ℃, every index through 18 months mensuration test strips all meets the demands, consider in transportation and the use that to the influence of test strips, we carry out 37 ℃ of accelerated tests of 7 days, experimental result shows that every index of test strips meets the requirements fully.
Sensitivity, specificity, accuracy and the stability that " magnetic immuno-chromatographic test paper strip of CA125 and kit in the detection by quantitative blood " is described is fully qualified.
Claims (7)
1. the magnetic immuno-chromatographic test paper strip of tumor-associated antigen 125 (CA125) in the detection by quantitative blood, it is characterized in that: this test strips is that interlaced successively 2mm ground is pasted coated film, the magnetic mat of particles that combines CA125 antibody, sample pad, adsorptive pads, covered the transparent plastic diaphragm seal on the upper strata and the test strips that is assembled into then on base plate, is coated with CA125 detection of antibodies line T and nature controlling line C on the coated film in advance.
2. according to the magnetic immuno-chromatographic test paper strip of CA125 in the detection by quantitative blood of claim 1, it is characterized in that: described sample pad is through the pretreated cellulose membrane of sample pad treating fluid, described sample pad treating fluid is to contain the polyvinyl alcohol (PVA) of 1%-5% casein and 0.1%-1% and the 0.02M of 0.01-0.2% Tween-20, the phosphate buffer of pH7.0-7.6.
3. according to the magnetic immuno-chromatographic test paper strip preparation method of CA125 in the detection by quantitative blood of claim 1, it is characterized in that: may further comprise the steps:
The preparation of A, fluorescein isothiocynate (FITC) antibody: select for use FITC to carry out the mark of CA125 antibody;
The preparation of B, magnetic particle: selecting diameter for use is the super paramagnetic particle of 100-300nm, the mode of using the carbodiimide covalent coupling with the anti-fluorescein antibody mark to the magnetic particle, the FITC-antibody that mark is good is with 1: 2-1: 5 molecule ratio (mol ratio) is mixed with the magnetic particle of coupling anti-fluorescein antibody, guarantees that the magnetic particle of coupling anti-fluorescein antibody is excessive;
C, use quantitative liquid-jet device to be sprayed on the magnetic mat of particles magnetic particle for preparing with the amount of 25 μ l/cm-50 μ l/cm;
The preparation of D, coated film: use 0.02mM, the PBS of pH7.0-7.6, respectively anti-CA 125 coated antibody and FITC-bovine serum albumin(BSA) are diluted to 0.5-2mg/ml concentration, use quantitative liquid-jet device respectively with the two with the interval spray printing of 0.5-1.0cm on nitrocellulose filter, dry the back and in confining liquid, soak after 10 minutes, add drying agent and seal up for safekeeping standby in 25-35 ℃ of oven dry 8 hours;
The processing of E, sample pad: sample pad is put into sample pad Treatment Solution immersion treatment take out 25-35 ℃ of oven dry 8 hours after 1 hour;
The assembling of F, test strips: interlaced successively 2mm sticks coated film on the transparent plastic base plate, the magnetic mat of particles, and sample pad, adsorptive pads covers the transparent plastic diaphragm seal then and obtains test paper plate on the upper strata, and the width cutting promptly obtains test strips as requested.
4. magnetic immuno-chromatographic standard measure according to claim 3 detects the preparation method of the test strips of CA125 in the blood, it is characterized in that described steps A is as follows:
With CA125 antibody 4 ℃ of dialysed overnight of PBS to 0.02M pH7.0-7.6, adjustment concentration is 2mg/ml, FITC is used the dimethyl formamide dissolving, final concentration is 50mmol, add the FITC solution of aequum with 5: 1 molecule ratios in antibody-solutions, room temperature reaction 1 hour is to 0.02M, 4 ℃ of dialysed overnight of the PBS of pH7.0-7.6 ,-20 ℃ of preservations are standby behind the adding equal-volume glycerine.
5. magnetic immuno-chromatographic standard measure according to claim 3 detects the preparation method of the test strips of CA125 in the blood, it is characterized in that described step B comprises following two steps:
1) preparation of the magnetic particle of coupling anti-fluorescein antibody: use the 50mmol that contains 0.1%Tween-20, the PBS washing magnetic particle of pH7.0-7.6, adding carbodiimide and succinimide makes the two final concentration be 20mmol, room temperature reaction 1 hour, fully adding anti-fluorescein antibody behind the washing magnetic particle, to make the molecular proportion of anti-fluorescein antibody and magnetic particle be 5: 1 (mol ratio), room temperature reaction 3 hours, add the 0.02M that contains 0.5%BSA, the PBS room temperature sealing of pH7.0-7.6 30 minutes, washing magnetic particle, use contains 1%PVP, 1%Casein, 0.5%Tween-20, the 50mmol of 5% sucrose, the boric acid of pH8.2-9.0 is preserved damping fluid, redissolution magnetic particle, and 4 ℃ of preservations are standby;
2) the magnetic particle of FITC-antibody and coupling anti-fluorescein antibody mixes, amount with 0.5 μ l/mg magnetic particle adds FITC-CA125 antibody in the magnetic particle solution of coupling anti-fluorescein antibody, fully use behind the mixing and preserve damping fluid with 1: 5-1: 20 ratio (volume ratio) is used mixture diluted fiberglass packing to be sprayed.
6. detect the preparation method of the test strips of CA125 in the blood according to the described magnetic immuno-chromatographic standard measure of claim 3, it is characterized in that: among the described step C, the spraying method of magnetic particle is: use quantitative spray film device evenly to be sprayed on the glass fibre with the amount of 50 μ l/cm the magnetic particle for preparing, add drying agent after the freeze drying and seal up for safekeeping standby.
7. detect the preparation method of the test strips of CA125 in the blood according to the described magnetic immuno-chromatographic standard measure of claim 3, it is characterized in that: among the described step D, the preparation method of coated film is: being cushioned liquid with bag is 0.5mg/ml with the CA125 antibody dilution, the FITC-BSA dilution is 1mg/ml, use quantitative spray film device with the amount of 1 μ l/cm with the two with the interval spray printing of 0.6cm on nitrocellulose filter, room temperature is dried after 30 minutes and to be soaked in confining liquid after 10 minutes in 25-35 ℃ of oven dry 8 hours, adds drying agent and seals up for safekeeping standby.
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