CN105717308A - Immunochromatography kit for fast and quantitatively detecting fecal lactoferrin - Google Patents
Immunochromatography kit for fast and quantitatively detecting fecal lactoferrin Download PDFInfo
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- CN105717308A CN105717308A CN201610172060.0A CN201610172060A CN105717308A CN 105717308 A CN105717308 A CN 105717308A CN 201610172060 A CN201610172060 A CN 201610172060A CN 105717308 A CN105717308 A CN 105717308A
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- lactoferrin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Abstract
The invention provides an immunochromatography kit for fast and quantitatively detecting fecal lactoferrin, and belongs to the technical field of clinic in-vitro diagnostic reagents. The immunochromatography kit comprises a test strip, the test strip comprises a bottom plate, a sample pad, a marker combining pad, a nitrocellulose membrane and a water absorbing pad, wherein the sample pad, the marker combined pad, the nitrocellulose membrane and the water absorbing pad are arranged on the bottom plate. The marker combined pad is coated with a lactoferrin monoclonal antibody A marked by colloidal gold particles, or colloidal silver particles, or colloidal iron particles, or magnetic particles, or fuel particles, or latex particles or fluorescence particles; the nitrocellulose membrane contains a detection line T and a quality control line C, the detection line T is formed by a lactoferrin monoclonal antibody B, and the quality control line C is formed by goat-anti-mouse IgG; the lactoferrin monoclonal antibody A and the lactoferrin monoclonal antibody B are used for recognizing different epitopes of lactoferrin. The immunochromatography kit has the advantages of being convenient, fast, easy to operate, accurate in result and suitable for fast clinic detection, and a preparing method is simple, efficient and low in cost.
Description
Technical field
The invention belongs to clinical in vitro diagnosis in vitro reagent technique field, be specifically related to the immune chromatography reagent kit of a kind of Quantitative detection feces lactoferrin.
Background technology
Irritable bowel syndrome (Irritable Bowelsyndrome, IBS) it is one group and accompanies the abnormal syndrome lacking organic disease foundation as feature of bowl evacuation habit change, feces character with stomachache and abdominal discomfort, it it is gastrointestinal dysfunction disease more typically, its main pathophysiological basis is digestive tract power how unrest, visceral hypersensitivity, brain-gut axis interaction imbalance etc., how based on dietary adjustments, symptomatic treatment and psychological intervention in treatment, prognosis is preferable.Inflammatory bowel (Inflammatory Bowel Disease,
IBD) burst carcinous colitis (ulcerativecolitis, UC are included
) and Crohn disease (Crohn's
Disease, CD), a kind of diarrhoea with recurrent exerbation, have blood in stool, generate heat, suffer from abdominal pain, the weight loss chronic intestinal disease as clinical manifestation, generally entail parenteral performance, pathological manifestations is gastrointestinal membranes and (or) the chronic inflammatory disease of gastrointestinal wall holostrome, its morbidity may cause body caused to the imbalance of Intestinal Antigens immunne response with the Different types of etiopathogenises such as heredity, environment and intestinal microbial population, aminosallcylic acid is needed in treatment, even glucocorticoid, immunosuppressant and biological preparation, and the state of an illness is prone to repeatedly, possible medication throughout one's life, threat to life time serious.
Typical IBD with IBS has nothing in common with each other in clinical manifestation, but Recent study finds, a lot of IBD patients show as IBS symptom such as abdominal distention, abdomen gulf, stomach discomfort etc. at the initial stage of a disease with clinical relieving period, according to the I diagnostic criteria of IBS Rome, IBS sample prodrome sustainable 2.7 years UC patient, CD patient sustainable 6.8 years, and in this stage, even if there being the guides such as existing rome criteria can want to accurately distinguish IBD Yu IBS or extremely difficult according only to Clinical presentation and feature for reference.But because pathomechanism each other, therapeutic modality and prognosis are entirely different, both Differential Diagnosis is challenge greatly for major part clinicist.
In clinical practice, it is typically based on the symptom of patient, sign and scope and laboratory examination results comprehensive descision IBD disease activity.Endoscopy combined lesion mucosa biopsy is the goldstandard of present stage assessment enteritis degree, can directly observe diseased region, specify extent of disease, degree and Histological change thereof, but have and check that the various parameters under invasive, time-consuming, scope and microscope are difficult to the shortcomings such as quantization；Although some hematological indices such as CRP, ESR, albumin, PLT counting, WBC counting etc. also can be used for evaluating IBD disease activity, but these indexs lack organ specificity, all can be significantly raised when each system inflammation of whole body, infection.Therefore, a considerable amount of IBS sample patient with sympotoms need to accept further invasive diagnosis and treatment such as endoscopy and radioactivity imaging examination with clear and definite whether be organic or functional illness, this not only adds the misery of patient and the risk (especially for the old seriously ill patient of child and part) of associative operation, and increase the weight of its financial burden, cause the waste of part medical resource.Clinical data shows, the Endoscopic patient of most acceptance is actual does not has serious organic disease, and the patient of chronic abdominal discomfort there is no need to carry out endoscopy at the medical initial stage.Thus, find a kind of Noninvasive, inexpensive safety, easy repeatable, again have significant for the Differential Diagnosis of IBD and IBS with specific laboratory parameters compared with hypersensitivity.
Lactoferrin (Lactoferrin
LF) be a kind of relative molecular mass be that the ferrum of 76-80kD combines glycoprotein, it is widely present in the external secretion such as milk, saliva or blood plasma, neutrophilic granulocyte secondary granule, is the main component of the M7 of activation, for a kind of product of inflammatory reaction.There is a large amount of neutrophil infiltration owing to being in the intestinal mucosa of inflammation, cause the rising of feces neutrophilic granulocyte derived protein (such as lactoferrin etc.) concentration, then measure these protein concentrations in feces and then can react enteritis situation the most reliably.
Feces lactoferrin (Fecal Lactoferrin, FLA) as the label of a kind of enteritis, can be used to differentiate struvite (including bacterial infection enteritis, activeness IBD) and non-inflammation intestinal tract disease, for diagnosis, course inflammatory activity degree and the assessment of curative effect of medication and the prediction of palindromia of enteritis.
At present, detection feces lactoferrin (Fecal Lactoferrin
, FLA) method be presently mainly Enzyme-multiplied immune technique (ELISA), elisa technique has the disadvantage in that detection equipment requirements is high, and cost is high；Interference factor is more, and repeatability is bad；The detection time is long.Therefore Enzyme-multiplied immune technique detection feces lactoferrin (Fecal Lactoferrin, FLA) is not suitable for clinical quick diagnosis.
Summary of the invention
It is an object of the present invention to provide the immune chromatography reagent kit of a kind of Quantitative detection feces lactoferrin, there is the advantages such as convenient and swift, simple to operate, result is accurate, be suitable to clinical quickly detection.
It is a further object of the present invention to provide the preparation method of mentioned reagent box, simply, efficiently, low cost.
The purpose of the present invention adopts the following technical scheme that realization.
A kind of immune chromatography reagent kit of Quantitative detection feces lactoferrin, including test strips, described test strips includes base plate, it is arranged at the sample pad on described base plate, label pad, nitrocellulose filter and adsorptive pads, described label pad is coated with colloid gold particle, colloidal silver particles, colloidal iron particles, magnetic-particle, fuel particle, lactoferrin monoclonal antibody A of latex particle or fluorescent particle markers, described nitrocellulose filter contains detection line T and nature controlling line C, described detection line T is formed by lactoferrin monoclonal antibody B, described nature controlling line C is formed by sheep anti-mouse igg；Described lactoferrin monoclonal antibody A and B identify the different epi-positions of lactoferrin.
Preferably in technical scheme, lactoferrin monoclonal antibody A is available from the Lactoferrin of Bo Mei bio tech ltd, Zhuhai
Monoclonal 13H1, production code member is M0153；Lactoferrin monoclonal antibody B is available from the Lactoferrin of Bo Mei bio tech ltd, Zhuhai
Monoclonal 5A4, production code member is M0154；Sheep anti-mouse igg is purchased from Hangzhou Long Ji Bioisystech Co., Ltd, and production code member is C56-Ab1.
Preferably in technical scheme, described sample pad, label pad, nitrocellulose filter and adsorptive pads overlap successively.
Preferably in technical scheme, described test kit also includes getting stuck, described get stuck include getting stuck and under get stuck, described upper and lower getting stuck connects and fixes, be provided with the locating slot for placing test strips in getting stuck under described, described on get stuck that the position corresponding to nitrocellulose membrane is provided with observation window, position corresponding to sample pad is provided with well.
Preferably in technical scheme, the material of described adsorptive pads is paper material.
Preferably in technical scheme, described base plate is coated with adhesive corresponding to the side of nitrocellulose filter, and the material of described base plate is polystyrene or polyethylene.
The present invention also provides for a kind of method preparing described test kit, comprises the steps:
(1) use surfactant buffer to soak glass fibre element film, be dried, obtain sample pad；
(2) use colloid gold particle, colloidal silver particles, colloidal iron particles, magnetic-particle, fuel particle, latex particle or fluorescent particle markers lactoferrin monoclonal antibody A, be then sprayed on glass fibre element film, be dried, obtain label pad；
(3) use spray film instrument lactoferrin monoclonal antibody B and sheep anti-mouse igg to be coated on respectively on nitrocellulose filter, form detection line T and nature controlling line C, obtain containing detection line T and the nitrocellulose filter of nature controlling line C；Described lactoferrin monoclonal antibody A and B identify the different epi-positions of lactoferrin；
(4) pasting sample pad, label pad, nitrocellulose filter and adsorptive pads on base plate, described sample pad, label pad, nitrocellulose filter and adsorptive pads overlap successively.
Preferably in technical scheme, the method using colloid gold particle labelling lactoferrin monoclonal antibody A is as follows: add wet chemical, stirring in colloidal gold solution；It is subsequently adding lactoferrin monoclonal antibody A, stirring；Add BSA aqueous solution, stir, be centrifuged, resuspended, obtain lactoferrin monoclonal antibody A of colloid gold particle labelling.
Preferably in technical scheme, described colloidal gold solution is adopted and is prepared with the following method: boiled by aqueous solution of chloraurate, adds trisodium citrate aqueous solution, heats, boil 5-15 minute, be cooled to room temperature, obtain colloidal gold solution under stirring under stirring.
Preferably in technical scheme, described surfactant buffer is to add the casein of final concentration of 4-6%, the polyvinylpyrrolidone of 4-6%, the sodium cholate of 0.5-1.5%, the RHODASURF of 1.0-2.0% in pH8.5-9.5 borate buffer
Formed after ON-870 and 0.01-0.03% sodium azide.
The immune chromatography reagent kit of Quantitative detection feces lactoferrin of the present invention, uses lateral chromatography immunological technique and immunoreation principle, detects feces lactoferrin by double antibody sandwich method.During inspection; first there is immunoreation with lactoferrin monoclonal antibody A of the colloid gold particle on label pad, colloidal silver particles, colloidal iron particles, magnetic-particle, fuel particle, latex particle or fluorescent particle markers in the lactoferrin antigen in sample, forms immune complex.Thereafter immune complex is along with sample chromatographic flow on nitrocellulose filter, when immune complex chromatographs the detection line T to nitrocellulose filter, reacts with lactoferrin monoclonal antibody B thus is fixed on the upper of nitrocellulose filter.Lactoferrin in fecal sample is the most, and the complex on detection line T is the most, and the optical density value on band is the highest.Meanwhile, during detection, lactoferrin monoclonal antibody A of free tape label is then combined with sheep anti-mouse igg at nature controlling line C and develops the color, the foundation the most sufficient as sample, test strips is the most working properly.
Due to colloid gold particle, colloidal silver particles, colloidal iron particles, magnetic-particle, fuel particle, latex particle or fluorescent grain at a particular wavelength; absorption to light is relevant to the amount of granule with scattering; therefore after reaction terminates; quantitative analysis instrument analysis detection line T and the optical density of nature controlling line C can be utilized, then according to the standard curve being set in advance in quantitative analysis instrument, the concentration of feces lactoferrin is calculated.
The present invention is by a large amount of creative work, have selected lactoferrin monoclonal antibody A, B and sheep anti-mouse igg and prepare the immune chromatography reagent kit of detection feces lactoferrin, the detection range of linearity of this test kit is 50ng/ml 3000ng/ml, convenient and swift, simple to operate, result is accurate, only need within 10 minutes, just can detect the concentration of feces lactoferrin in sample, drastically increase detection efficiency.The present invention is using granules such as gold colloidals as label, and this label has good stability, and is conducive to improving detection stability.The present invention is at the well being provided with sample interpolation and the observation window for observed result of getting stuck, according to analytical tool result of determination, accurately and reliably.The present invention is easy to make, and volume is little, be easy to carry.Testing cost of the present invention is relatively low, and good stability breaks away from cold chain, easily preserves, and easily transports, and can preserve for a long time (24 months), and beneficially grass-roots unit promotes.The present invention can be mass, it is adaptable to clinical quick diagnosis and on-the-spot quick diagnosis.Test kit of the present invention is simple to operate, convenient and swift, by intelligent immunity quantitative analysis instrument or automated stool analyser, result can be carried out interpretation, can realize automatization, reduces the impact of subjective factors, it is provided that convenient, quickly, reliably, diagnostic result accurately.
Accompanying drawing explanation
Fig. 1 is the structural representation of test strips in test kit of the present invention, wherein 1: sample pad, 2: label pad, 3: nitrocellulose filter, 4: adsorptive pads, 5: base plate.
Fig. 2 is the front view of the immune chromatography reagent kit of Quantitative detection feces lactoferrin of the present invention, wherein, 1: sample pad, 2: label pad, 3: nitrocellulose filter, 4: adsorptive pads, 5: base plate, 6: get stuck, 7: well, 8: observation window.
Fig. 3 is the top view of the immune chromatography reagent kit of Quantitative detection feces lactoferrin of the present invention, wherein, 6: get stuck, 7: well, 8: observation window, 9: detection line T, 10: nature controlling line C.
Detailed description of the invention
Below with reference to embodiment and accompanying drawing, the technique effect of design, concrete structure and the generation of the present invention is clearly and completely described, to be completely understood by the purpose of the present invention, feature and effect.Obviously; described embodiment is a part of embodiment of the present invention rather than whole embodiment, based on embodiments of the invention; other embodiments that those skilled in the art is obtained on the premise of not paying creative work, belong to the scope of protection of the invention.It addition, all connection/annexations being previously mentioned in literary composition, the most singly refer to that component directly connects, and refer to couple auxiliary according to being embodied as situation by adding or reducing, form more excellent draw bail.
The composition of the immune chromatography reagent kit of embodiment 1 detection by quantitative feces lactoferrin
Structure in conjunction with the immune chromatography reagent kit of-3 pairs of detection by quantitative feces lactoferrin of Fig. 1 is described.This immune chromatography reagent kit includes test strips, sample pad 1, label pad 2, nitrocellulose filter 3 and the adsorptive pads 4 that test strips includes base plate 5, is arranged on base plate 5, label pad 2 is coated with lactoferrin monoclonal antibody A of colloid gold particle labelling, nitrocellulose filter 3 is containing parallel detection line T and nature controlling line C, detection line T is formed by lactoferrin monoclonal antibody B, nature controlling line C is formed by sheep anti-mouse igg, and lactoferrin monoclonal antibody A, B identify the different epi-positions of lactoferrin.
Lactoferrin monoclonal antibody A is available from the Lactoferrin of Bo Mei bio tech ltd, Zhuhai
Monoclonal 13H1, is murine antibody, and production code member is M0153；Lactoferrin monoclonal antibody B is available from the Lactoferrin of Bo Mei bio tech ltd, Zhuhai
Monoclonal 5A4, production code member is M0154；Sheep anti-mouse igg is purchased from Hangzhou Long Ji Bioisystech Co., Ltd, and production code member is C56-Ab1.
Sample pad 1, label pad 2, nitrocellulose filter 3 and adsorptive pads 4 overlap successively.
Test kit also includes getting stuck 6, get stuck 6 include getting stuck and under get stuck, upper and lower getting stuck connects and fixes, under get stuck in be provided with the locating slot for placing test strips, the position corresponding to nitrocellulose membrane of above getting stuck is provided with observation window 8, position corresponding to sample pad is provided with well 7.
Observation window 8 approximate ellipsoidal.
The material of adsorptive pads is paper material.Base plate is coated with adhesive corresponding to the side of nitrocellulose filter, and the material of base plate is polystyrene or polyethylene.
The preparation of the immune chromatography reagent kit of embodiment 2 detection by quantitative feces lactoferrin
In embodiment 1, test kit is adopted and is prepared with the following method:
1. sample pad
Using surfactant buffer to soak glass fibre element film, at 37 DEG C, relative humidity is dried under the conditions of being less than 20%, obtains sample pad.Surfactant buffer is to add final concentration of 5%(mass percentage concentration in pH9.0 borate buffer) casein, 5%(mass percentage concentration) PVP-10(Polyvinylpyrrolidone 10), 1%(mass percentage concentration) sodium cholate, 1.8%(mass percentage concentration) RHODASURF
ON-870 (SIGMA company reagent), 0.02%(mass percentage concentration) sodium azide after formed.
PH9.0 borate buffer: add 980 milliliters of ultra-pure waters in 6.18 grams of boric acid and dissolve, then with 10 mol/L NaOH aqueous solutions tune pH to 9.0, adds ultra-pure water to 1000 milliliters of borate buffers i.e. obtaining pH9.0.
2. label pad
In 10mL, 1%(mass percentage concentration) in aqueous solution of chloraurate, add in 90mL ultra-pure water, heat under stirring, be rapidly added 14mL, 1%(mass percentage concentration after boiling) trisodium citrate aqueous solution, heat under stirring, close heater after boiling 10 minutes, under stirring, be cooled to room temperature (18～25 DEG C), obtain colloidal gold solution.
Take colloidal gold solution 100mL, add the wet chemical of 1mL, 0.1mol/L, stir 30 minutes.Add 0.1mg lactoferrin monoclonal antibody A, continue stirring 30 minutes.Add 1mL, 10%(mass percentage concentration) BSA(bovine serum albumin) aqueous solution, stir 30 minutes.At 4 DEG C, under the conditions of 12000rpm centrifugal 30 minutes, remove supernatant, with containing 1%(mass percentage concentration) the resuspended precipitation of 0.01mol/L, pH7.0 sodium phosphate buffer of BSA, obtain lactoferrin monoclonal antibody A of colloid gold particle labelling.With gold spraying instrument, lactoferrin monoclonal antibody A of colloid gold particle labelling is sprayed on glass fibre element film, at 37 DEG C, the relative humidity oven dried overnight less than 20%, obtains label pad.Lactoferrin monoclonal antibody A is available from the Lactoferrin Monoclonal 13H1 of Bo Mei bio tech ltd, Zhuhai, is murine antibody, and production code member is M0153.
3. nitrocellulose filter
With 0.01M, pH7.4 phosphate buffer (PBS), lactoferrin monoclonal antibody B is configured to the solution of 1.0-2.0mg/ml, rules with the parameter of 1.0 μ l/cm on nitrocellulose filter with spray film instrument, be coated detection line T.Then, the sheep anti-mouse igg of 0.5-1.5mg/ml uses spray film instrument rule on nitrocellulose filter with the parameter of 1.0 μ l/cm, is coated nature controlling line C.It is dried, obtains containing detection line T and the nitrocellulose filter of nature controlling line C.Detection line T and nature controlling line C is parallel to each other.Nitrocellulose filter be divide into three sections by detection line T and nature controlling line C, including part and nature controlling line C side between detection line T side, detection line T and nature controlling line C.
Lactoferrin monoclonal antibody B is available from the Lactoferrin Monoclonal 5A4 of Bo Mei bio tech ltd, Zhuhai, and production code member is M0154.Sheep anti-mouse igg is purchased from Hangzhou Long Ji Bioisystech Co., Ltd, and production code member is C56-Ab1；Lactoferrin monoclonal antibody A identifies the different epi-positions of lactoferrin from lactoferrin monoclonal antibody B.
4. adsorptive pads and base plate
Adsorptive pads is the paper material that water absorbing capacity is stronger.Baseboard material is polystyrene or polyethylene, is coated with the adhesive for pasting sample pad etc..
5. the assembling of test kit
Under conditions of relative humidity is less than 30%, take base plate, nitrocellulose filter 3 containing detection line T and nature controlling line C is pasted onto the middle part of base plate, at the detection line T side binding mark thing pad 2 of nitrocellulose filter, pastes sample pad 1 at label pad 2 opposite side；Adsorptive pads 4 is pasted in the nature controlling line C side of nitrocellulose filter 3.Sample pad, label pad, nitrocellulose filter and adsorptive pads overlap successively.Each adhesive portion mutually laminates l-2mm, and the big plate pasted is cut into the wide test strips of 4mm.Then test strips is respectively placed in down in the locating slot got stuck, covers and get stuck, compress, complete the assembling of test kit.
The using method of the immune chromatography reagent kit of embodiment 3 detection by quantitative feces lactoferrin and effect
By 200 parts of clinical fecal sample stool sample box samplings, add normal saline dilution mixing.In the well 7 of embodiment 1 test kit, dropping 3-4 drips the sample after process, after 10 minutes, utilize intelligent immunity quantitative analysis instrument or automated stool analyser analysis detection line T and the optical density of nature controlling line C, the concentration of feces lactoferrin in sample after processing according to the standard curve calculating being set in advance in analyser, unit is ng/ml.Calculating lactoferrin quality in every gram of fecal sample, unit is ug/g.
Above-mentioned 200 parts of clinical samples are used U.S. TECHLAB simultaneously
Inc. company produces
LACTOFERRIN SCAN test kit (ELISA) detects, and analyzes the dependency of two kinds of method testing results.Result shows that the feces lactoferrin concentration dependency that two kinds of detection methods obtain is fine, correlation coefficient r=0.9875, P > 0.05, no difference of science of statistics between two kinds of methods.Can be determined that the present invention's is functional by result above, there is the advantages such as easy and simple to handle, reaction is quick, result is the most credible, applicable Site Detection.
Through testing inspection, embodiment 1 test kit is 50ng/ml 3000ng/ml to the range of linearity of feces lactoferrin detection after processing, and is 5ug/g 300ug/g to the detection range of lactoferrin in fecal sample.
Although the detailed description of the invention to the present invention gives detailed description and explanation above, but it should be noted that, those skilled in the art need not too much test and can implement the present invention is required for protection, the example gold colloidal substitute being similar to: colloidal silver particles, colloidal iron particles, magnetic-particle, fuel particle, latex particle, or fluorescent grain replaces colloid gold particle or transforms, or the reagent of present invention description is replaced with some reagent similar on physics and chemistry, or carry out various equivalence change and amendment, function produced by it still without departing from description and accompanying drawing contained spiritual time, all should be within protection scope of the present invention.
Claims (10)
1. the immune chromatography reagent kit of a Quantitative detection feces lactoferrin, it is characterized in that: include test strips, described test strips includes base plate, it is arranged at the sample pad on described base plate, label pad, nitrocellulose filter and adsorptive pads, described label pad is coated with colloid gold particle, colloidal silver particles, colloidal iron particles, magnetic-particle, fuel particle, lactoferrin monoclonal antibody A of latex particle or fluorescent particle markers, described nitrocellulose filter contains detection line T and nature controlling line C, described detection line T is formed by lactoferrin monoclonal antibody B, described nature controlling line C is formed by sheep anti-mouse igg；Described lactoferrin monoclonal antibody A and B identify the different epi-positions of lactoferrin.
The immune chromatography reagent kit of Quantitative detection feces lactoferrin the most according to claim 1, it is characterized in that: lactoferrin monoclonal antibody A is available from the Lactoferrin Monoclonal 13H1 of Bo Mei bio tech ltd, Zhuhai, production code member is M0153；Lactoferrin monoclonal antibody B is available from the Lactoferrin of Bo Mei bio tech ltd, Zhuhai
Monoclonal 5A4, production code member is M0154；Sheep anti-mouse igg is purchased from Hangzhou Long Ji Bioisystech Co., Ltd, and production code member is C56-Ab1.
The immune chromatography reagent kit of Quantitative detection feces lactoferrin the most according to claim 2, it is characterised in that: described sample pad, label pad, nitrocellulose filter and adsorptive pads overlap successively.
The immune chromatography reagent kit of Quantitative detection feces lactoferrin the most according to claim 3, it is characterized in that: described test kit also includes getting stuck, described get stuck include getting stuck and under get stuck, described upper and lower getting stuck connects and fixes, be provided with the locating slot for placing test strips in getting stuck under described, described on get stuck that the position corresponding to nitrocellulose membrane is provided with observation window, position corresponding to sample pad is provided with well.
5. according to the immune chromatography reagent kit of Quantitative detection feces lactoferrin described in claim 1 or 2 or 3 or 4, it is characterised in that: the material of described adsorptive pads is paper material.
The immune chromatography reagent kit of Quantitative detection feces lactoferrin the most according to claim 5, it is characterised in that: described base plate is coated with adhesive corresponding to the side of nitrocellulose filter, and the material of described base plate is polystyrene or polyethylene.
7. the method preparing the described test kit of one of claim 1-6, it is characterised in that: comprise the steps:
(1) use surfactant buffer to soak glass fibre element film, be dried, obtain sample pad；
(2) use colloid gold particle, colloidal silver particles, colloidal iron particles, magnetic-particle, fuel particle, latex particle or fluorescent particle markers lactoferrin monoclonal antibody A, be then sprayed on glass fibre element film, be dried, obtain label pad；
(3) use spray film instrument lactoferrin monoclonal antibody B and sheep anti-mouse igg to be coated on respectively on nitrocellulose filter, form detection line T and nature controlling line C, obtain containing detection line T and the nitrocellulose filter of nature controlling line C；Described lactoferrin monoclonal antibody A and B identify the different epi-positions of lactoferrin；
(4) pasting sample pad, label pad, nitrocellulose filter and adsorptive pads on base plate, described sample pad, label pad, nitrocellulose filter and adsorptive pads overlap successively.
The method preparing test kit the most according to claim 7, it is characterised in that: the method using colloid gold particle labelling lactoferrin monoclonal antibody A is as follows: add wet chemical, stirring in colloidal gold solution；It is subsequently adding lactoferrin monoclonal antibody A, stirring；Add BSA aqueous solution, stir, be centrifuged, resuspended, obtain lactoferrin monoclonal antibody A of colloid gold particle labelling.
The method preparing test kit the most according to claim 8, it is characterized in that: described colloidal gold solution is adopted and prepared with the following method: boiled by aqueous solution of chloraurate, add trisodium citrate aqueous solution, heat under stirring, boil 5-15 minute, under stirring, it is cooled to room temperature, obtains colloidal gold solution.
10. according to the described method preparing test kit of one of claim 7-9, it is characterised in that: described surfactant buffer is to add formation after the casein of final concentration of 4-6%, the polyvinylpyrrolidone of 4-6%, the sodium cholate of 0.5-1.5%, the RHODASURF ON-870 of 1.0-2.0% and 0.01-0.03% sodium azide in pH8.5-9.5 borate buffer.
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| CN110208544A (en) * | 2019-06-20 | 2019-09-06 | 南方医科大学深圳医院 | A kind of excrement Test paper and kit |
| CN110244062A (en) * | 2019-07-18 | 2019-09-17 | 珠海市医友生物科技有限公司 | A kind of excrement calprotectin or lactoferrin combined detection reagent and preparation method thereof |
| CN111896749A (en) * | 2020-08-07 | 2020-11-06 | 杭州都林生物科技有限公司 | Bovine lactoferrin double-antibody sandwich colloidal gold immunochromatographic test strip, preparation method, kit and detection method |
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| CN109100505A (en) * | 2018-08-22 | 2018-12-28 | 宁波奥丞生物科技有限公司 | A kind of immunofluorescent reagent box of quantitative detection blood vessel endothelium marker CD105 |
| 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 |
| CN110208544A (en) * | 2019-06-20 | 2019-09-06 | 南方医科大学深圳医院 | A kind of excrement Test paper and kit |
| CN110244062A (en) * | 2019-07-18 | 2019-09-17 | 珠海市医友生物科技有限公司 | A kind of excrement calprotectin or lactoferrin combined detection reagent and preparation method thereof |
| CN111896749A (en) * | 2020-08-07 | 2020-11-06 | 杭州都林生物科技有限公司 | Bovine lactoferrin double-antibody sandwich colloidal gold immunochromatographic test strip, preparation method, kit and detection method |
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