CN105785041A - Test strip for quantitatively detecting calprotectin, preparation method thereof and determining method for calprotectin concentration - Google Patents
Test strip for quantitatively detecting calprotectin, preparation method thereof and determining method for calprotectin concentration Download PDFInfo
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Abstract
The invention discloses a test strip for quantitatively determining calprotectin, a preparation method thereof and a determining method for calprotectin concentration, belonging to the technical field of biotechnology. The test strip comprises a base plate, wherein a sample absorbing pad, a labeled antibody pad, a wrapped film and a water absorbing pad are successively arrayed and attached to the base plate. The test strip provided by the invention has the advantages of accurate test result, convenience and low cost.
Description
Technical field
The present invention relates to biological technical field, be specifically related to the instant rapid assay methods of a kind of immuno-chromatographic test paper strip for detection by quantitative people's calprotectin (calprotectin) and preparation method thereof and calprotectin (calprotectin) concentration.
Background technology
Calprotectin (Calprotectin) is molecular weight is the calcium of 36kD, zinc-binding protein.The calcium-binding protein heterotrimer connected with covalent bond by the light chain that the heavy chain that two molecular weight are 14kD and molecular weight are 8kD forms.
Calprotectin is considered as reliable inflammation index in multiple disease, and it can clearly distinguish organic disease such as diseases associated with inflammation IBD and functional disease such as IBS.This in-vitro diagnosis instrument can avoid expense and the invasive inspection of colonoscope of costliness.Gastroenterology association of Britain (BSG) annual meeting that 14-17 day in March, 2005 holds in Birmingham, seminar inflammatory bowel (IBD)-calprotectin-irritable bowel syndrome (IBS), using calprotectin as a new useful diagnosis reference mark, it is used for: difference inflammatory bowel and irritable bowel syndrome；Assessment inflammatory bowel inflammatory activities；Assessment inflammatory bowel mucosa Cure.
The detection of the calprotectin of feces is a kind of simple, rapid, highly sensitive, high specificity, inflammatory bowel detection method cheap, noninvasive.This detection method all can obtain testing result accurately in the Mucositis of the mucosa of normal person and afflicted patient.Receiver operating curve's (ROC curve) analyzes and shows that the point of cut-off of every 150ug/g just can distinguish inflammatory bowel (inflammatoryboweldisease, and irritable bowel syndrome (irritablebowelsyndrome IBD), IBS) patient, its sensitivity is 100%, and specificity has reached 97%.
For the detection of calprotectin, method clinical at present includes: the methods such as euzymelinked immunosorbent assay (ELISA) (ELISA), colloidal gold method, but these methods have respective feature and deficiency.ELISA method is owing to quantitatively accurately, being widely used in hospital laboratory, but the detection time is long, not convenient；Although colloidal gold method is convenient, fast, but being a kind of qualitative method, its sensitivity is relatively low；Colloid gold particle and antibody connected mode belong to Electrostatic Absorption, come off result can be caused inaccurate, unstable in detection process.
Therefore, urgent needs sets up a kind of quick, convenient, economic calprotectin quantitative detecting method clinically.
Summary of the invention
In view of this, the embodiment of the present invention provides a kind of immuno-chromatographic test paper strip for detection by quantitative calprotectin, and it has with low cost, measures advantage easily.
The preparation method that present invention also offers a kind of immuno-chromatographic test paper strip for detection by quantitative calprotectin.
Present invention also offers the application of a kind of immuno-chromatographic test paper strip for detection by quantitative calprotectin.
On the one hand, the present invention provides a kind of test strips for detection by quantitative calprotectin, and described test strips includes base plate, and described base plate is attached with the sample absorption pad, traget antibody pad, coated film and the adsorptive pads that are longitudinally arranged in order.
Further, described sample absorption pad, traget antibody pad, coated film and adsorptive pads are from left to right arranged in order on described base plate, and the length ratio of described sample absorption pad, traget antibody pad, coated film and adsorptive pads is 13-17:10-12:20-24:24-24.
Further, described traget antibody pad includes the first calprotectin monoclonal antibody being coated with label.
Further, the first calprotectin monoclonal antibody of described label labelling is fluorescently-labeled anti-calprotectin antibody.
Further, described coated film being coated with detection band and quality control band, described detection band is fixed with the second calprotectin monoclonal antibody, and described nature controlling line is fixed with rabbit anti-mouse igg antibody.
Further, described coated film is nitrocellulose filter.
On the other hand, the preparation method that the present invention provides a kind of test strips for detection by quantitative calprotectin, comprise the steps: on the base plate of test strips, fix sample absorption pad, traget antibody pad, coated film and adsorptive pads successively.
Further, described coated film is prepared via a method which and obtains:
Use the first phosphate buffer to dilute rabbit anti-mouse IgG respectively and anti-calprotectin antibody obtains rabbit anti-mouse IgG solution and anti-calprotectin antibody solution, the first described phosphate buffering liquid concentration is 0.01-0.03M, pH value is 7.2-7.6, and described rabbit anti-mouse IgG solution and the concentration of anti-calprotectin antibody solution are 1mg/ml-1.5mg/ml；Use quantitatively spray film instrument to be sprayed on nitrocellulose filter on by the two with the interval of 0.5cm-1.0cm with the amount of 0.8-1.2 μ l/cm, dry 0.5-1.5h for 35-38 DEG C, add desiccant and seal up for safekeeping standby.
Further, described traget antibody pad is prepared by the following method and is obtained:
Prepare anti-calprotectin antibody solution: with the first borate buffer solution, anti-calprotectin antibody dialysis is obtained anti-calprotectin antibody solution, the first described borate buffer solution concentration is 0.03-0.07M, pH value is 8.0-8.5, and dialysis temperature is 3-5 DEG C, dialysis time T >=16h；Described anti-calprotectin antibody solution concentration is adjusted to 1.5-2mg/ml；
Fluorescent microsphere pretreatment: use the MES activation buffer washing fluorescent microsphere of pH4.5-5.0, add carbodiimide and butanimide makes the two final concentration be 18-22mmol/L, room temperature reaction 10-20min, fully wash fluorescent microsphere；
Add in described anti-calprotectin antibody solution after the fluorescent microsphere described in pretreated being redissolved with the second borate buffer solution that the pH value that concentration is 0.03-0.07M is 8.0-8.5, the mass ratio making described anti-calprotectin antibody and fluorescent microsphere is 1-4: 50, room temperature reaction 1.5-2.5h, add the second phosphate buffer room temperature and close 20-40min, in the second described phosphate buffer, the mass fraction of BSA is 10%, the second described phosphate buffered solution concentration is 0.02M, and pH value is 7.2-7.6；Redissolve to reacting precursor long-pending with triphosphate buffer preserving liquid, described triphosphate buffer includes the Tween-20 of BSA and the 0.2-0.3% of 0.3-0.7% by mass percentage, described triphosphate pH of cushioning fluid is 7.2-7.6, and concentration is 0.01-0.03M；Use quantitatively spray film instrument to be sprayed on fiberglass packing with 3-5 μ l/cm, dry 1h for lucifuge 35-38 DEG C, add desiccant and seal up for safekeeping standby.
Another further aspect, the present invention also provides for the assay method of a kind of calprotectin concentration, comprises the steps:
The drafting of standard curve: calprotectin standard substance are configured to gradient concentration standard solution and carry out fluorescent strength determining, with detect line, nature controlling line fluorescence intensity ratio for vertical coordinate, calprotectin concentration of standard solution is abscissa, fits to standard curve.
The detection of sample: dripped to by testing sample in test strips, then detects with fluorescence detection device, draws the concentration of calprotectin in described sample；
Described test strips is the test strips described in any one of claim 1-6.
During use, sample pad adds sample liquid, under capillary action, sample liquid is to the swimming of adsorptive pads one end, when in specimen to be measured containing calprotectin (calprotectin), calprotectin forms complex with the antibodies on fluorescent microsphere, along with chromatography effect, complex moves forward arrival detection line T place, calprotectin in complex and the anti-calprotectin antibody on coated film combine and are gathered in T line place, unconjugated Fluorescent microsphere marker may proceed to move ahead, when arriving nature controlling line C, rabbit anti-mouse IgG antibody mouse monoclonal antibody on fluorescent microsphere is combined, the gathering of fluorescent microsphere occurs at C line place.Whole reaction completed in 15 minutes, and carried out upper machine-read card, and T line and C line all can produce corresponding fluorescence signal, and fluorescence detector can calculate quantitative result according in the standard curve that the substitution of actually detected value is preset by the information on calibration card.
Compared with prior art, the present invention at least has the advantage that
The present invention by calprotectin monoclonal antibody covalent coupling on fluorescent microsphere, as the mobile phase of detection after being sprayed on fiberglass packing, rabbit anti-mouse IgG and another kind of calprotectin monoclonal antibody are coated on nitrocellulose filter as catching solid phase, conventionally immunochromatography ratio juris carries out the detection of specimen, detect in conjunction with simple and easy to do fluorescence detector, while realizing high-sensitivity detection, the time of detection can be greatly shortened again.
By the improvement to test strips, fluorescence immune chromatography technology is introduced in the detection of calprotectin, combined with fluorescent detector, it is achieved that single part detection by quantitative of calprotectin, provide great convenience for Clinical practice.
The present invention is easy and simple to handle, be suitable for large-scale production, and the portable set needed for detection also lists, and the quantitative Diagnosis for calprotectin has positive meaning.
Calprotectin is detected, the present patent application achieves the innovation in the detection system being different from above method: can be greatly shortened again the time of detection while high-sensitivity detection calprotectin, single part detection by quantitative is realized in 15 minutes, provide great convenience for Clinical practice, be more suitable for the operation of specialty section office.
Accompanying drawing explanation
Fig. 1 is the Facad structure schematic diagram of calprotectin test strip of the present invention；
Fig. 2 is the side structure schematic diagram of calprotectin test strip of the present invention；
Fig. 3 is the examination criteria curve in the embodiment of the present invention 3；
The fecal calprotectin that Fig. 4 is the application present invention and BuhlmannLaboratoriesAG company of Switzerland defends protein detection kit (euzymelinked immunosorbent assay (ELISA)), the dependency diagram to the calprotectin concentration value that 40 example samples measure.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in further detail, it will be appreciated that following example are to facilitate those skilled in the art's understanding to the present invention program, but not as a limitation of the invention.
A kind of immuno-chromatographic test paper strip for detection by quantitative calprotectin, described test strips includes base plate, and described base plate is attached with the sample absorption pad, traget antibody pad, coated film and the adsorptive pads that are arranged in order.
Test strips in such scheme can complete detection, provides preferred version herein below on basis:
Described sample absorption pad, traget antibody pad, coated film and adsorptive pads are from left to right arranged in order on described base plate, and the length ratio of described sample absorption pad, traget antibody pad, coated film and adsorptive pads is 13-17:10-12:20-24:20-24；Described traget antibody pad includes the first calprotectin monoclonal antibody being coated with label；First calprotectin monoclonal antibody of described label labelling is the anti-calprotectin antibody of fluorescent labeling or colloid gold label；Being coated with detection band and quality control band on described coated film, described detection band is fixed with the second calprotectin monoclonal antibody, and described nature controlling line is fixed with rabbit anti-mouse igg antibody；Described coated film is nitrocellulose filter.
The preparation method of above-mentioned test strips comprises the steps: to fix sample absorption pad, traget antibody pad, coated film and adsorptive pads on the base plate of test strips successively；
Described coated film is prepared via a method which and obtains:
Use the first phosphate buffer to dilute rabbit anti-mouse IgG respectively and anti-calprotectin antibody obtains rabbit anti-mouse IgG solution and anti-calprotectin antibody solution, the first described phosphate buffering liquid concentration is 0.01-0.03M, pH value is 7.2-7.6, and described rabbit anti-mouse IgG solution and the concentration of anti-calprotectin antibody solution are 1mg/ml-1.5mg/ml；Use quantitatively spray film instrument to be sprayed on nitrocellulose filter on by the two with the interval of 0.5cm-1.0cm with the amount of 0.8-1.2 μ l/cm, dry 0.5-1.5h for 35-38 DEG C, add desiccant and seal up for safekeeping standby.
Described traget antibody pad is prepared by the following method and is obtained:
Prepare anti-calprotectin antibody solution: with the first borate buffer solution, anti-calprotectin antibody dialysis is obtained anti-calprotectin antibody solution, the first described borate buffer solution concentration is 0.03-0.07M, pH value is 8.0-8.5, and dialysis temperature is 3-5 DEG C, dialysis time T >=16h；Described anti-calprotectin antibody solution concentration is adjusted to 1.5-2mg/ml；
Fluorescent microsphere pretreatment: use the MES activation buffer washing fluorescent microsphere of pH4.5-5.0, add carbodiimide and butanimide makes the two final concentration be 18-22mmol/L, room temperature reaction 10-20min, fully wash fluorescent microsphere；
Add in described anti-calprotectin antibody solution after the fluorescent microsphere described in pretreated being redissolved with the second borate buffer solution that the pH value that concentration is 0.03-0.07M is 8.0-8.5, the mass ratio making described anti-calprotectin antibody and fluorescent microsphere is 1-4: 50, room temperature reaction 1.5-2.5h, add the second phosphate buffer room temperature and close 20-40min, in the second described phosphate buffer, the mass fraction of BSA is 10%, the second described phosphate buffered solution concentration is 0.02M, and pH value is 7.2-7.6；Redissolve to reacting precursor long-pending with triphosphate buffer preserving liquid, described triphosphate buffer includes the Tween-20 of BSA and the 0.2-0.3% of 0.3-0.7% by mass percentage, described triphosphate pH of cushioning fluid is 7.2-7.6, and concentration is 0.01-0.03M；Use quantitatively spray film instrument to be sprayed on fiberglass packing with 3-5 μ l/cm, dry 1h for lucifuge 35-38 DEG C, add desiccant and seal up for safekeeping standby.
Specific embodiment is presented herein below
Embodiment 1
The preparation of the fluorescence immune chromatography test paper bar of calprotectin:
A, antibody preparation: select commercial calprotectin monoclonal antibody, with 4 DEG C of dialysed overnight (18h) of borate buffer solution of 0.05MpH8.5；Select commercial calprotectin monoclonal antibody, by the PBS4 DEG C of dialysed overnight (16h) of 0.02MpH7.4；
B, labelling calprotectin monoclonal antibody the preparation of fluorescent microsphere pad: fluorescent microsphere (the Bangslab company selecting diameter to be 100nm, excitation wavelength 350nm, detection wavelength 615nm), with 0.05M, it is mass fraction 1% that pH4.5MES buffer regulates microsphere concentration, then uses the mode of carbodiimide (EDC) and butanimide (NHS) covalent coupling by calprotectin labeling of monoclonal antibody to fluorescent microsphere.The fluorescent microsphere prepared use quantitatively spray film instrument be sprayed on fluorescent microsphere pad with the amount of 3 μ l/cm；
C, nitrocellulose filter preparation:
The pH7.4PBS that concentration is 0.02M containing mass fraction 1% sucrose is used respectively calprotectin monoclonal antibody and rabbit anti-mouse IgG antibody to be all diluted to 1mg/ml concentration, quantitatively spray film instrument is used to be sprayed on nitrocellulose filter by the two with the interval of 0.5cm respectively, dry 1h for 35-38 DEG C, add desiccant and seal up for safekeeping standby；
The process of D sample pad:
Sample pad is put into immersion treatment in sample pad treatment fluid (the 0.1MpH7.4 phosphate buffer containing mass fraction 1%BSA and 0.1%TritonX-100 or the 0.1MTris buffer containing mass fraction 1%BSA) and, after 1 hour, dries 5h for 35-38 DEG C；
The assembling of E test strips:
Operations described below in humidity less than 38%, must carry out in the room of temperature 20-25 DEG C.
On PVC base plate successively nitrocellulose filter in interlaced 2mm stickup, combine the fluorescent microsphere pad of anti-calprotectin antibody, sample pad, adsorptive pads, be then cut into 0.6cm width, namely obtain test strips.
Fig. 1 is the Facad structure schematic diagram of test strips in the present invention, and Fig. 2 is the side structure schematic diagram of test strips in the present invention；As depicted in figs. 1 and 2, the fluorescence immune chromatography test paper bar of the calprotectin of the present embodiment includes being coated with quality control band C and detecting the nitrocellulose filter 4 with T, be covered in the high-intensity water absorbent paper 4 of nitrocellulose filter side, the fluorescently-labeled calprotectin monoclonal antibody pad being covered in the opposite side of nitrocellulose filter and sample pad 1 on base plate 6, base plate；Described detection band T is coated with calprotectin monoclonal antibody；Described quality control band is coated with rabbit anti-mouse igg antibody.
Embodiment 2
The test strips preparation method of the present embodiment comprises the following steps:
A, antibody preparation: select commercial calprotectin monoclonal antibody, by 4 DEG C of dialysed overnight of borate buffer solution of 0.05MpH8.5；Select commercial calprotectin monoclonal antibody, by the PBS4 DEG C of dialysed overnight of 0.02MpH7.4；
B, labelling calprotectin monoclonal antibody the preparation of colloidal gold pad:
Take gold colloidal 50ml, filter.
Dilute with water antibody 3 times (about 1mg/ml), adds in 30min, just mixes while add, and near ice, mixing dynamics is little, it is prevented that destroy the connection of antibody and gold colloidal
Add 10%BSA, make solution final concentration of 1%, add 10%PEG and make final concentration of 0.2%, add in 15min.
10000r/min, 60min are centrifugal, careful sucking-off supernatant, the precipitate PB liquid (containing 0.02%NaN3) containing 1%BSA, will precipitate resuspended 1/10,4 DEG C of preservation for original volume.
The colloid gold label thing prepared use quantitatively spray film instrument be sprayed in label pad with the amount of 3 μ l/cm.
C, nitrocellulose filter preparation:
The 0.02MpH7.4PBS containing 1% sucrose is used respectively calprotectin monoclonal antibody and rabbit anti-mouse IgG antibody to be all diluted to 1mg/ml concentration, quantitatively spray film instrument is used to be sprayed on nitrocellulose filter by the two with the interval of 0.5cm respectively, dry 1h for 35-38 DEG C, add desiccant and seal up for safekeeping standby；
The process of D sample pad:
Sample pad is put into immersion treatment in sample pad treatment fluid (the 0.1MpH7.4 phosphate buffer containing 1%BSA and 0.1%TritonX-100 or the 0.1MTris buffer containing 1%BSA) and, after 1 hour, dries 5h for 35-38 DEG C；
The assembling of E test strips:
Operations described below in humidity less than 38%, must carry out in the room of temperature 20-25 DEG C.
On PVC base plate successively nitrocellulose filter in interlaced 2mm stickup, combine the colloidal gold pad of anti-calprotectin antibody, sample pad, adsorptive pads, be then cut into 0.6cm width, namely obtain test strips.
As depicted in figs. 1 and 2, the colloidal gold immuno-chromatography test paper strip of the calprotectin of the present embodiment includes being coated with quality control band C and detecting the nitrocellulose filter 4 with T, be covered in the high-intensity water absorbent paper 5 of nitrocellulose filter side, the calprotectin monoclonal antibody pad being covered in the colloid gold label of the opposite side of nitrocellulose filter and sample pad 1 on base plate 6, base plate；
Described detection band T is coated with calprotectin monoclonal antibody；Described quality control band is coated with rabbit anti-mouse igg antibody.
Embodiment 3
Calprotectin (calprotectin) concentration in fluorescence immune chromatography detection by quantitative feces
1. the drafting of standard curve
Calprotectin standard substance are configured to 600ug/g, 300ug/g, 100ug/g, 30ug/g, 10ug/g, 0ug/g, and by the test strips of same batch, each point is tested 6 times.According to statistical method, with detect line (T band), nature controlling line (C band) fluorescence intensity ratio (T band detected value/C band detected value) for vertical coordinate, calprotectin (calprotectin) concentration of standard solution is abscissa, setting up equation and fit to standard curve, standard curve is as shown in Figure 3.
2. the detection of sample:
1) with inoculating loop, 50-100mg fecal sample is put in test tube, add 49 times of sample net weight Extraction buffer (containing 2.5M carbamide, 0.025MCaCl2,0.25M citric acid, 0.5% sodium azide, 1.25%BSA the 0.25MTris buffer of pH8.0).Mixing, room temperature is placed 10 minutes, centrifugal 5 minutes of 3000g, takes supernatant and is sample extraction thing.
2) taking out the detector bar of embodiment 1 from packing box, after tearing packaging of aluminium foil bag, keep flat detector bar, take 100 μ l sample extraction things and add in well, room temperature lucifuge is reacted 15 minutes.
3) opening fluorescence detection device, and detector bar and calibration card insert the card inserting mouth of fluorescence detection device, run instrument, instrument calculates the concentration of the calprotectin in sample to be tested automatically by corresponding software of analyzing.
It is currently known calprotectin mensuration and only has applicable hospital laboratory for the euzymelinked immunosorbent assay (ELISA) (ELISA) of batch detection, chemiluminescence (CLIA), but without being suitable for single part, quickly detecting, immediately go out the detection of result.
Calprotectin is detected, this patent achieves the innovation in the detection system being different from above method: can be greatly shortened again the time of detection while high-sensitivity detection calprotectin, the single part detection by quantitative realized, provides great convenience for Clinical practice, is more suitable for the operation of specialty section office.
3) defend the dependency that protein detection kit (euzymelinked immunosorbent assay (ELISA)) detects to compare with the fecal calprotectin of BuhlmannLaboratoriesAG company of Switzerland.
Adopt strip and the detection system of fluorescence detection device composition of the present invention, have detected 40 example human serum samples according to above-mentioned detection method；Meanwhile, defend protein detection kit (euzymelinked immunosorbent assay (ELISA)) with the fecal calprotectin of BuhlmannLaboratoriesAG company of Switzerland and detect 40 above-mentioned example samples.Testing result is in Table 1, and dependency compares sees Fig. 4.The dependency r > 0.975 of two kinds of method testing results, has significance.
Defend protein detection kit (euzymelinked immunosorbent assay (ELISA)) with the fecal calprotectin of BuhlmannLaboratoriesAG company of Switzerland and detect the testing result of 40 above-mentioned example samples for abscissa, the result measured with the method for the present patent application, for vertical coordinate, draws correlation curve；Result shows that the two dependency is fine.
Table 1. this law is defended protein detection kit (euzymelinked immunosorbent assay (ELISA)) detection and is compared with the fecal calprotectin of BuhlmannLaboratoriesAG company of Switzerland
The present invention program does not use up part, and those skilled in the art can select existing technology to complete as required, such as the concrete size of test strips, and concrete time that detection is used and sample taken amount etc..
Above example is only the exemplary embodiment of the present invention, is not used in the restriction present invention, and protection scope of the present invention is defined by the claims.The present invention in the essence of the present invention and protection domain, can be made various amendment or equivalent replacement by those skilled in the art, and this amendment or equivalent replacement also should be regarded as being within the scope of the present invention.
Claims (10)
1. the test strips for detection by quantitative calprotectin, it is characterised in that described test strips includes base plate, and described base plate is attached with the sample absorption pad, traget antibody pad, coated film and the adsorptive pads that are longitudinally arranged in order.
2. the test strips for detection by quantitative calprotectin according to claim 1, it is characterized in that, described sample absorption pad, traget antibody pad, coated film and adsorptive pads are from left to right arranged in order on described base plate, and the length ratio of described sample absorption pad, traget antibody pad, coated film and adsorptive pads is 13-17:10-12:20-24:24-24.
3. the described test strips for detection by quantitative calprotectin according to claim 1, it is characterised in that described traget antibody pad includes the first calprotectin monoclonal antibody being coated with label.
4. the described test strips for detection by quantitative calprotectin according to claim 3, it is characterised in that the first calprotectin monoclonal antibody of described label labelling is the anti-calprotectin antibody of fluorescent labeling or colloid gold label.
5. the described test strips for detection by quantitative calprotectin according to claim 1, it is characterized in that, being coated with detection band and quality control band on described coated film, described detection band is fixed with the second calprotectin monoclonal antibody, and described nature controlling line is fixed with rabbit anti-mouse igg antibody.
6. the immuno-chromatographic test paper strip for detection by quantitative calprotectin according to claim 1, it is characterised in that described coated film is nitrocellulose filter.
7. the preparation method of the test strips for detection by quantitative calprotectin described in a claim 1-6 any one, it is characterised in that fix sample absorption pad, traget antibody pad, coated film and adsorptive pads on the base plate of test strips successively.
8. the preparation method of the test strips for detection by quantitative calprotectin according to claim 7, it is characterised in that described coated film is prepared via a method which and obtains:
Use the first phosphate buffer to dilute rabbit anti-mouse IgG respectively and anti-calprotectin antibody obtains rabbit anti-mouse IgG solution and anti-calprotectin antibody solution, the first described phosphate buffering liquid concentration is 0.01-0.03M, pH value is 7.2-7.6, and described rabbit anti-mouse IgG solution and the concentration of anti-calprotectin antibody solution are 1mg/ml-1.5mg/ml；Use quantitatively spray film instrument to be sprayed on nitrocellulose filter on by the two with the interval of 0.5cm-1.0cm with the amount of 0.8-1.2 μ l/cm, dry 0.5-1.5h for 35-38 DEG C, add desiccant and seal up for safekeeping standby.
9. method according to claim 7, it is characterised in that described traget antibody pad is prepared by the following method and obtained:
Prepare anti-calprotectin antibody solution: with the first borate buffer solution, anti-calprotectin antibody dialysis is obtained anti-calprotectin antibody solution, the first described borate buffer solution concentration is 0.03-0.07M, pH value is 8.0-8.5, and dialysis temperature is 3-5 DEG C, dialysis time T >=16h；Described anti-calprotectin antibody solution concentration is adjusted to 1.5-2mg/ml；
Fluorescent microsphere pretreatment: use the MES activation buffer washing fluorescent microsphere of pH4.5-5.0, add carbodiimide and butanimide makes the two final concentration be 18-22mmol/L, room temperature reaction 10-20min, fully wash fluorescent microsphere；
Add in described anti-calprotectin antibody solution after the fluorescent microsphere described in pretreated being redissolved with the second borate buffer solution that the pH value that concentration is 0.03-0.07M is 8.0-8.5, the mass ratio making described anti-calprotectin antibody and fluorescent microsphere is 1-4: 50, room temperature reaction 1.5-2.5h, add the second phosphate buffer room temperature and close 20-40min, in the second described phosphate buffer, the mass fraction of BSA is 10%, the second described phosphate buffered solution concentration is 0.02M, and pH value is 7.2-7.6；Redissolve to reacting precursor long-pending with triphosphate buffer preserving liquid, described triphosphate buffer includes the Tween-20 of BSA and the 0.2-0.3% of 0.3-0.7% by mass percentage, described triphosphate pH of cushioning fluid is 7.2-7.6, and concentration is 0.01-0.03M；Use quantitatively spray film instrument to be sprayed on fiberglass packing with 3-5 μ l/cm, dry 1h for lucifuge 35-38 DEG C, add desiccant and seal up for safekeeping standby.
10. the assay method of a calprotectin concentration, it is characterised in that comprise the steps:
The drafting of standard curve: calprotectin standard substance are configured to gradient concentration standard solution and carry out fluorescent strength determining, with detect line, nature controlling line fluorescence intensity ratio for vertical coordinate, calprotectin concentration of standard solution is abscissa, fits to standard curve.
The detection of sample: dripped to by testing sample in test strips, then detects with fluorescence detection device, draws the concentration of calprotectin in described sample；
Described test strips is the test strips described in any one of claim 1-6.
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| CN107085116A (en) * | 2017-05-16 | 2017-08-22 | 张子林 | It is a kind of to detect kit of calprotectin and preparation method thereof in human faecal mass sample |
| CN107782894A (en) * | 2016-08-24 | 2018-03-09 | 北京怡成生物电子技术股份有限公司 | Avoid the test card of influence of moisture and its application during accuracy tested person |
| CN108333368A (en) * | 2018-02-07 | 2018-07-27 | 深圳市伯劳特生物制品有限公司 | The kit and preparation method of calprotectin in a kind of detection human faecal mass |
| CN109682979A (en) * | 2019-01-30 | 2019-04-26 | 珠海市银科医学工程股份有限公司 | A kind of calprotectin joint lactoferrin antigen test strip and preparation method thereof |
| WO2021057988A1 (en) * | 2019-09-27 | 2021-04-01 | 廖睿 | Use of reagent for detecting content of faecal calprotectin in preparation of kit for screening fallopian tube lesions |
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| WO2021057988A1 (en) * | 2019-09-27 | 2021-04-01 | 廖睿 | Use of reagent for detecting content of faecal calprotectin in preparation of kit for screening fallopian tube lesions |
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Application publication date: 20160720 |