CN108267592A - A kind of immune microsphere chromatography detects heparin-binding protein Test paper - Google Patents
A kind of immune microsphere chromatography detects heparin-binding protein Test paper Download PDFInfo
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- CN108267592A CN108267592A CN201710000257.0A CN201710000257A CN108267592A CN 108267592 A CN108267592 A CN 108267592A CN 201710000257 A CN201710000257 A CN 201710000257A CN 108267592 A CN108267592 A CN 108267592A
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- G—PHYSICS
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- 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
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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Abstract
The present invention relates to the technical fields of clinical infectious disease marker detection, and in particular to a kind of immune microsphere chromatography detection heparin-binding protein Test paper.Include the following steps：It is prepared by calibration curve；The preparation of detection buffer solution containing rabbit-anti people's heparin-binding protein antibody, the preparation of the chicken anti-rabbit secondary antibody of fluorescence or color latex microballoon label, sample pad coating solid phase, the reacting pad preparation for being coated with the anti-human heparin-binding protein antibody detection line of mouse and being coated with goat anti-rabbit antibody nature controlling line, seperation film assembling, the assembling of test strips, clinical sample detection, and dry test strips are placed in aluminium foil bag.Fluorescent chromatographic method detection heparin-binding protein Test paper of the present invention has higher cost benefit, and quantify level of the detect and assess heparin-binding protein in the body fluid of patient.This method is for POCT detecting systems and screening or detects the variation of heparin-binding protein in patient body fluid to determine the detection method to infecting marker, and heparin-binding protein detection technique can be made to become routine clinical detection project.
Description
Technical field
The present invention relates to the biomarker heparin-binding protein inspection technology field of infectious diseases more particularly to one kind
Immune microsphere chromatography detects heparin-binding protein Test paper preparation method.
Background technology
HBP has higher sensitivity and specificity as a kind of marker of inflammation than Procalcitonin.Because HBP is main
It is to be discharged by PMN by environmental stimuli, so HBP contents are very low in normal person's blood, is usually no more than 10ng/mL, when there is infection
During generation, part bacterium invades intravascular, and thalline is in itself or the material incentives neutrophil leucocyte such as toxin of bacterium release is released
HBP is put to increase so as to cause HBP contents in blood.HBP can reach 20~30ng/mL in nonspecific infection, and severe infections can in ICU
It can exceed that 100ng/mL is even as high as more than 1000ng/mL；When HBP contents be more than 1000ng/mL when patient be in extreme
In danger, it is faced with possible dead danger at any time.Just because of HBP plays the role of so powerful, just necessary it is being faced
Using progress numerous studies in bed monitoring and treatment.
At present, clinically using HBP >=15ng/mL as the mark of Severe sepsis, sensibility, specificity, the positive are pre-
Measured value and negative predictive value can reach 87.1%, 95.1%, 88.4% and 94.5% respectively；In more than index, calcium drops
The indexs such as plain raw water is put down, IL-6 levels, lactoferrin level, CRP levels, quantity of leucocyte may have outstanding performance in a certain item,
But other are very low, and even as low as 30% or so.Severe sepsis often leads to the generation of circulatory failure, therefore, is generating heat
In patient, the height of blood HBP contents is whether prediction patient can develop into circulatory failure selection indicators.
Pyemia is to lead to the major reason of critical patient death, and its illness rate is still rising in recent years, early diagnosis
For reducing case fatality rate, it is most important to improve prognosis.It is well known that it is to cause that Coagulation Dysfunction, systemic inflammatory response are unbalance
Organ dysfunction or even the major reason of failure during pyemia.Research shows that inflammatory reaction early stage, neutrophils chemotactic,
Pathophysiological process caused by migration, activation is the initiating agent for causing endothelial cell damage, is speculated accordingly, HBP may be in purulence
It plays an important role in the occurrence and development of toxication.
HBP mainly by neutrophil leucocyte it is stimulated when be released into blood, human normal plasma HBP concentration is very low, is usually no more than
The horizontal significantly raising of 10ng/mL, patients with septic shock blood plasma HBP, HBP can be used for the hair of prediction shock and circulatory failure
It is raw.Linder etc. thinks that blood plasma HBP levels are more than the best laboratory index that 15ng/mL is severe sepsis diagnosis, sensibility
Up to 87.1%, specificity 95.1%, positive predictive value 88.4%, negative predictive value 94.5%.Akesson etc. has found pyemia group
Blood plasma HBP levels are apparently higher than control group, dynamic detection HBP levels can auxiliary judgment pyemia lapse to, HBP levels and disease are tight
Weight degree is related, and high HBP levels increase death risk.Before there is blood pressure reduction, HBP levels have risen sepsis patient
Height finds the investigation of 338 patients with severe sepsis 143 patients only have HBP raising tables before organ failure occurs
It is existing, wherein 80% blood plasma HBP concentration is more than 30ng/mL, it is average to occur organ failure after 10.5h.The discoveries such as Huang Airong, HBP
It is respectively to the sensitivity of prediction children with sepsis, specificity, positive predictive value, negative predictive value higher than 6.79ng/mL
84.21%th, 84.09%, 69.57% and 92.5%, joint-detection HBP and remaining alkali can improve specificity.In this regard, we also need
It more to study and verify its clinical value.
In conclusion HBP is a kind of granule protein of neutrophil leucocyte release, generates releasing mechanism and also need to further
Research.HBP has the advantages that highly sensitive and specificity as clinical emerging Inflammatory Mediators, how by HBP and tradition inflammation
Marker combines, to find the indication of quicker, special, sensitive diagnosis of sepsis disease, it is still necessary to further exploratory development.With
It and HBP researchs is goed deep into, it is reason to believe that HBP will be used as quite valuable biomarker in diagnosis of sepsis
Play the effect of bigger.
The HBP detection reagents that western countries use at present are mainly ELISA method, and this method is in external clinical clinical research
In it is very universal, it is complicated for operation but due to the testing staff for needing profession, it is long to expend the time, it is impossible to be detected by bed.Sweden simultaneously
Lufthansa medicine is using HBP as the application patent of diagnosis septicemia biomarker, and using HBP levels as the diagnosis of urinary tract infections
The diagnostic method patent of process patent, meningitis, but the said firm does not have specific diagnostic reagent method.Axis-
Shield companies have registered heparin-binding protein ELISA method kit in CFDA, but since ELISA method detects heparin knot
Hop protein clinical manipulation is complicated, and the consuming time is long, and clinic cannot be popularized, and CN204882574U, which discloses heparin-binding protein, to be determined
Detection kit is measured, the patent is using colloidal gold immunochromatographimethod, and there are Stability and veracities because of colloidal gold immunochromatographimethod
Not high limitation, market in urgent need is a kind of more accurate, and more sensitive detection method is for heparin-binding protein detection by bed.Hangzhou
The patent of immunofluorescence chromatography detection heparin-binding protein has been applied in middle Han Shengtai biologies and the raw medical treatment of Henan life
CN204882575U and CN105572386A, but not by fluorescent labeled antibody solid phase in patent, clinical manipulation is cumbersome, together
When patent CN204882575U increase an auxiliary detection line, there are the instable hidden danger of testing result.
Clinical limitation during heparin-binding protein detection is carried out for above method, we provides a kind of immune microsphere
Chromatography detects heparin-binding protein Test paper, the test strip application fluorescent microsphere or dyed microspheres label secondary antibody, and
Will label secondary antibody solid-phase coating in sample pad, avoid the unstability of conventional tag antibodies buffer, improve detection sensitivity,
The HBP markers in blood sample are quantitatively detected by immune lateral chromatography technology.
Invention content
(1) technical problems to be solved
It is accurately, reliably, quantitative the technical problem to be solved in the present invention is to provide sensitive, it is at low cost, quickly, hold
A kind of immune microsphere chromatography detection heparin-binding protein Test paper preparation method easily used, using fluorescent staining label two
Anti- particle is as instruction system, and solid-phase coating is in sample pad, to replace existing colloid gold label and fluorescent marker detection side
Method improves label detection stability, quantitatively detects heparin-binding protein in sample, and lateral immune chromatography method can be used for patient's
Bed is other to be detected, and compensates for the limitation of laboratory ELISA method detection.
(2) technical solution
In order to solve the above technical problem, the present invention provides a kind of detection heparin-binding protein inspections of immune microsphere chromatography
Test paper preparation method, Fig. 1 are the schematic diagram of the test strips of the present invention.
A kind of immune microsphere chromatography detection heparin-binding protein Test paper preparation method provided by the invention is included such as
Lower step：
Position 1 shown in figure one is the heparin-binding protein detected in sample, which is detected by immunochromatography, passed through
It prepares heparin-binding protein standard curve and determines the HBP concentration levels of unlike signal value, by rabbit-anti people HBP antibody-HBP- mouse
Anti-human HBP antibody (sandwich method) quantitatively detects HBP levels；Key step is prepared including calibration curve；Contain rabbit-anti people's heparin knot
The preparation of the chicken anti-rabbit secondary antibody of the preparation of the detection buffer solution of hop protein antibody, fluorescence or color latex microballoon label, sample pad
Coating solid phase is coated with the anti-human heparin-binding protein antibody detection line of mouse and is coated with the reacting pad system of goat anti-rabbit antibody nature controlling line
Standby, seperation film assembling, the assembling of test strips, clinical sample detection, and dry test strips are placed in aluminium foil bag.The present invention is glimmering
Light chromatography detection heparin-binding protein Test paper has higher cost benefit, and quantify detect and assess heparin-binding protein
Level in the body fluid of patient.For realization more than technical purpose, this invention takes following technical schemes：
Prepared by S1, calibration curve, choose heparin-binding protein standard items, and the dilution of employment standard blank serum is configured to following
Different concentration：0.1ug/L、0.5ug/L、1ug/L、5ug/L、10ug/L、50ug/L、100ug/L、150ug/L、200ug/L、
250ug/L for the detection heparin-binding protein Test paper detection of immune microsphere chromatography, demarcates the detection range of batch test paper
Calibration curve；
S2, containing heparin-binding protein antibody detection buffer solution preparation, position 2 as shown in Figure 1, wherein it is preferred that but
Be not limited to prepare PH6.7-7.4 phosphate buffers, add in protein protective agent, carbohydrate, surfactant, preservative one kind or
Several formulated in combination detect buffer solution, and antibody sources include but not limited to rabbit-anti people, the anti-human heparin-binding protein antibody of mouse, antibody
Concentration is including but not limited to 1ng/ul-3ng/ul；
The preparation of secondary antibody that S3, microballoon mark, position 4 as shown in Figure 1, wherein being preferably but not limited to prepare PH6.7-
7.4 phosphate buffers add in the one or more combination preparation label of seralbumin, carbohydrate, surfactant, preservative
Buffer solution preferably marks secondary antibody with the fluorescence of carboxylated or color latex microballoon, and antibody concentration is including but not limited to 0.1ug/ul-
3ug/ul；
S4, sample pad coating solid phase, position 4 as shown in Figure 1, wherein it is preferred that applying but being not limited to glass fibre membrane, nitre
Acid cellulose film, cellulose acetate film, NC films, with one or more of preservatives, surfactant, seralbumin, sodium chloride,
Carbohydrate reagent is impregnated, and the label secondary antibody solid phase in step S3 is sprayed at sample pad, is placed in 37-56 DEG C of drying process, storage
It is stored in sealing bags for use；
Prepared by S5, the anti-human heparin-binding protein antibody detection line of coating mouse, position 5 as shown in Figure 1, wherein preferred antibody
Package amount includes but not limited to 0.1-1ug/cm, and antibody coating buffer solution includes one or more of preservatives, surfactant, blood
Pure albumen, sodium chloride, carbohydrate reagent combination, are placed in 37-56 DEG C of drying process；
S6, the preparation of goat anti-rabbit antibody nature controlling line, position 6 as shown in Figure 1 are coated with, wherein it is preferred that preparing goat anti-rabbit antibody
Control liquid is coated with nature controlling line according to the amount of 0.1-2ug/cm；
S7, the assembling of seperation film and test strips, the assembling of test strips, position 3 as shown in Figure 1 is seperation film, wherein excellent
Choosing will mark the sample pad of secondary antibody to be assembled into viscose carrier plastics plate one end in S3, firmly presses, will be coated with nature controlling line
It is assembled in the middle part of test strips with the film of detection line, the other end of test strips pastes absorbing membrane, sample lateral chromatography is maintained, by test paper
Item is put into plastic casing, is placed on plastic box bottom by one section of sample pad, plastic casing top is fixed in blotting paper one end；
S8, clinical sample detection, according to heparin-binding protein standard curve, at room temperature the sample pad position of test strips according to
It is secondary to add in detection buffer solution and clinical samples, quantitatively detect heparin binding protein white level；
The heparin-binding protein calibration curve range in step sl, judges different infection indexs, wherein seriously
HBP concentration in septic patient sample is preferably greater than 20ng/ml, for the HBP concentration in meningitis clinical samples
Preferably greater than 50ng/ml, 50ng/ml is preferably greater than for HBP concentration in the clinical samples of urinary tract infections.
The clinical samples need including but not limited to blood and urine according to infection detection in step sl, wherein sternly
Weight septic patient and meningitis clinical samples generally use whole blood and ingredient blood sample, and the clinical samples of urinary tract infections are general
HBP level detections are carried out with urine.
The buffer solution added in step s 2 includes but not limited to PBS, HEPES, Tris buffer solution, wherein being preferably
The PBS buffer solution of 10mM PH6.7-7.4；The glucide of addition includes but not limited to add in sucrose, trehalose, glucose etc.
Carbohydrate, glucide additional proportion include but not limited to 1%-10%, wherein preferably 5% sucrose；The serum of addition is white
Albumen includes but not limited to BSA, HSA, standard serum, and ratio includes but not limited to 1%-10%, wherein preferably 5%BSA.
2nd, described a kind of immune microsphere chromatography detection heparin-binding protein Test paper preparation method, wherein microballoon or
Colour developing grain diameter includes but not limited to 0.1-1 microns, preferably 0.3um microballoons.
3rd, it is micro- that the microballoon or colour developing particle, wherein microballoon or colour developing particle include but not limited to fluorescent microsphere, colour
Ball, preferably fluorescent microsphere.
4th, sample pad coating solid phase, wherein sample pad are coated with the secondary antibody of microballoon label, which can be same
When can combine detection buffer solution in heparin-binding protein antibody and reacting pad nature controlling line coated antibody.
5th, the reaction pad film includes but not limited to nitrocellulose filter, cellulose acetate film.
6th, a kind of immune microsphere chromatography detection heparin-binding protein Test paper, the test paper testing result need to insert
The immune quantitative analyzer of Ru Lepu companies reads the fluorescent value or OD value of sample to be tested.Wherein preservative in sample pad
Including but not limited to biological preservatives such as Sodium azide, thimerosal, gentamicin sulphate and combinations thereof.
(3) advantageous effect
The above-mentioned technical proposal of the present invention has the advantages that：The present invention detects liver for a kind of immune microsphere chromatography
The preparation method of plain binding protein Test paper using the fluorescence or dyed microspheres of secondary antibody label as instruction system, dyes micro-
The covalent bond secondary antibody of ball and HBP antibody, with the conventional colloidal gold of substitution and ELISA detection HBP methods, microballoon label secondary antibody solid phase
Sample pad simplifies the clinical manipulation of the clinical Quantitative detection of HBP, improve on existing market ELISA detection it is sensitive
Degree, stability, accuracy quantitatively detect HBP by POCT fluorescence or optical density detector, prompt different infectious diseases
HBP gradeds, easy to operate when making clinical examination, time saving, sample dosage is few, generally only needs 50ul clinical samples,
Testing result is clearly accurate, and repeatability is strong, and testing result achievees the effect that naked eyes and the accurate interpretation of automatic machinery, inspection simultaneously
Result is surveyed to be easy to standardize.Control nature controlling line is increased when carrying out the experiment of HBP Test papers simultaneously, ensure experimental result has
Effect property and quality control.
Description of the drawings
Fig. 1 is the schematic diagram that a kind of immune microsphere chromatography of the embodiment of the present invention detects heparin-binding protein Test paper；
Fig. 2 is implementation steps flow chart of the present invention
Specific embodiment
Embodiments of the present invention are described in further detail with reference to the accompanying drawings and examples.Following embodiment is used for
Illustrate the present invention, but cannot be used for limiting the scope of the invention.
In the description of the present invention, unless otherwise indicated, " multiple " are meant that two or more.Term " upper ", "
Under ", " left side ", " right side ", " interior ", " outside ", " front end ", " rear end ", " head ", " " it is base to wait the orientation of instructions or position relationship for tail portion
In orientation shown in the drawings or position relationship, it is for only for ease of the description present invention and simplifies description rather than instruction or imply
Signified device or element must have specific orientation, with specific azimuth configuration and operation, therefore it is not intended that this
The limitation of invention.In addition, term " first ", " second ", " third " etc. are only used for description purpose, and it is not intended that instruction or dark
Show relative importance.
In the description of the present invention, it should be noted that unless otherwise clearly defined and limited, term " installation ", " phase
Even ", " connection " should be interpreted broadly, for example, it may be being fixedly connected or being detachably connected or be integrally connected；It can
To be mechanical connection or be electrically connected；It can be directly connected, can also be indirectly connected by intermediary.For this
For the those of ordinary skill in field, the concrete meaning of above-mentioned term in the present invention can be understood with concrete condition.
Embodiment one
As shown in Figure 1, a kind of immune microsphere chromatography detection heparin-binding protein Test paper preparation method, including as follows
Step：
S1, calibration curve prepare specific steps：
Specific steps：It is female that 5ug/ml heparin-binding proteins are prepared with normal person's standard serum (being purchased from Jiangsu En Moasai)
Liquid, dilute mother liquor heparin-binding protein standard items, heparin-binding protein diluted concentration for 0.1ug/L, 0.5ug/L, 1ug/L,
5ug/L, 10ug/L, 50ug/L, 100ug/L, 150ug/L, 200ug/L, 250ug/L are matched with heparin-binding protein Test paper
Close POCT immunity analysis instrument demarcation signal values and concentration curve.
Embodiment two
S2, the preparation for detecting buffer solution containing heparin-binding protein antibody
Specific steps：Rabbit-anti people's heparin-binding protein antibody (biocare) is prepared with the PBS buffer solution of PH7.4,10mM,
Compound concentration is 2ng/ul, adds in 5% sucrose, 5% BSA, 0.1% Tween-20,0.05% Sodium azide.
Embodiment three
The preparation of secondary antibody that S3, microballoon mark
Specific steps：The fluorescent microsphere of the 0.3um of 100ml 10% is taken, is washed 3 times with the MES solution of PH4.5 50mM,
The EDAC solution (MES of PH4.550mM is prepared) of the 10mg/ml of 1ml Fresh is added in, is incubated at room temperature 30min, Ran Houyong
2000rpm centrifuges 5min, is then washed with the MES solution of PH4.550mM, with chicken anti-rabbit secondary antibody (PBS) room of the 1ug/ul of 1ml
Temperature is incubated overnight, and is incubated at room temperature 1 hour with 10%BSA, 100 × Tris-EDTA, is washed 3 times with 1mlPBS after centrifugation, and storage is used
In 4% sucrose, 0.05% sodium azide solution.
Example IV
S4, sample pad coating solid phase
Specific steps：The membrane derived Millipore of glass fibre, with the ethylenediamine tetraacetic (propoxylation-embedding for containing 1% of mixing
Section-ethoxylate) tetrol surfactant, 0.8% casein and 5 × Tris-EDTA immersions, 56 DEG C of dryings, drying
Afterwards, secondary antibody microballoon of the volume of mixture than 2.5% marked, 5% sucrose, 2.5% trehalose, 5 × Tris-EDTA,
Using professional spray gun, in height 5-7mm with the secondary antibody of 75mm seconds spraying microballoon labels of speed, 56 DEG C of dried for standby.
Embodiment five
It is prepared by S5, the anti-human heparin-binding protein antibody detection line of coating mouse
Specific steps：The anti-human heparin-binding protein antibody (biocare) of mouse, compound concentration are prepared with PH7.4,10mM PBS
3ug/ml adds in 5% sucrose, 5% BSA, 0.1% Tween-20,0.05% Sodium azide.It is sprayed with the constant speed of 45mm/s
Painting forms p-wire.
Embodiment six
S6, the preparation of goat anti-rabbit antibody nature controlling line is coated with,
Specific steps：Will include 0.25mg/mL goat-anti rabbit heparin-binding protein antibody (biocare) reference reagent with
1% sucrose/0.5% trehalose/10mM TAPS pH 9.0 are mixed, and nature controlling line was sprayed under 0.075 μ l/mm with 45mm/ seconds.
S7, the assembling of seperation film and test strips, the assembling of test strips
Specific steps choose the high-quality glass fibre membrane BT100 of Millipore as sample cushion material, cut into 30 ×
2cm sizes are spare, choose 30 × 2cm blotting papers, and specification is the PVC plastic flitch of 30 × 6cm, is stained with double faced adhesive tape, then by NC films, sample
Product pad, blotting paper are pasted in PVC board, and the lap of splice between the film of each specification is no more than 0.5mm, convenient for lateral chromatography,
The test strips of 4mm wide are cut into after assembling, in packing and aluminium foil bag, 4 DEG C of storages.
S8, clinical sample detection,
Specific steps：It is sequentially added at the sample pad position of test strips at room temperature containing rabbit-anti people's heparin-binding protein antibody
Detection buffer solution, clinical samples add in patient's difference body fluid according to various disease, and general pyemia, meningitis and septicemia are suffered from
Person is detected with whole blood sample, and patients of urinary tract infection is detected with urine specimen, coordinates POCT immune quantitative quantitative analyses
Instrument detects heparin binding protein white level；
In conclusion the present invention provides a kind of immune microsphere chromatography detection heparin-binding protein Test paper preparation sides
Method, the fluorescence or dyed microspheres of this method application label secondary antibody are as instruction system, the covalent label secondary antibody and HBP of microballoon
Antibody, with the conventional colloidal gold of substitution and ELISA method detection HBP, the clinical rapid fluorescence for solving HBP is quantitatively detected, is improved
The sensitivity of colloidal gold ELISA method detection on existing market, stability, accuracy are detected by POC instrument quantitatives
The level of HBP prompts weak, stronger, the strong graded of level of HBP, and easy to operate when making clinical examination, time saving, sample is used
Amount is few, generally only needs 50ul clinical samples, and testing result is clearly accurate, and repeatability is strong, and testing result reaches simultaneously
Naked eyes and the effect of the accurate interpretation of automatic machinery, testing result are easy to standardize.Simultaneously in the horizontal detection test paper for carrying out HBP
Control nature controlling line is increased during experiment, ensures validity and the quality control of experimental result.
The embodiment of the present invention provides for the sake of example and description, and is not exhaustively or by this to send out
It is bright to be limited to disclosed form.Many modifications and variations are obvious for the ordinary skill in the art.Choosing
It is to more preferably illustrate the principle of the present invention and practical application to select and describe embodiment, and makes those of ordinary skill in the art
It will be appreciated that the present invention is so as to design the various embodiments with various modifications suitable for special-purpose.
Claims (6)
1. a kind of immune microsphere chromatography detects heparin-binding protein Test paper, can in clinical assisted diagnosis patient heparin
Binding protein is horizontal, and including the use of instant immunochromatography, quantitatively or in qualitative determination patient the body fluid levels of heparin-binding protein are
It is no to be higher than preset range, which is characterized in that include the following steps：
Prepared by S1, calibration curve, because heparin-binding protein index judges in different body fluid the concentration level of different infection, to heparin
Binding protein detection range is demarcated, it is characterised in that the standard solution of calibration including but not limited to 0.1ug/L, 0.5ug/L,
1ug/L、5ug/L、10ug/L、50ug/L、100ug/L、150ug/L、200ug/L、250ug/L；
S2, containing heparin-binding protein antibody detection buffer solution preparation, it is characterised in that with including but not limited to buffer solution,
The one or more combination preparation of protein protective agent, carbohydrate, surfactant, preservative detects buffer solution, and antibody sources include
But rabbit-anti people, the anti-human heparin-binding protein antibody of mouse are not limited to, antibody concentration is including but not limited to 1ng/ul-3ng/ul；
The preparation of secondary antibody that S3, microballoon mark, it is characterised in that with including but not limited to buffer solution, seralbumin, carbohydrate, table
The one or more combination preparation label buffer solution of face activating agent, preservative, with carboxylated or amidized fluorescence or colored breast
Glue microballoon marks secondary antibody, and antibody concentration is including but not limited to 0.1ug/ul-3ug/ul；
S4, sample pad coating solid phase, it is characterised in that apply but be not limited to glass fibre membrane, nitrocellulose filter, acetate fiber
Plain film, NC films, are impregnated with one or more of preservatives, surfactant, seralbumin, sodium chloride, carbohydrate reagent, will
Label secondary antibody solid phase in step S3 is sprayed at sample pad, is placed in 37-56 DEG C of drying process, is stored in sealing bags and treats
With；
Prepared by S5, the anti-human heparin-binding protein antibody detection line of coating mouse, it is characterised in that antibody package amount includes but not limited to
0.1-1ug/cm, antibody coating buffer solution include one or more of preservatives, surfactant, seralbumin, sodium chloride, sugar
Class reagent combines, and is placed in 37-56 DEG C of drying process；
S6, it is coated with the preparation of goat anti-rabbit antibody nature controlling line, it is characterized in that goat anti-rabbit antibody control liquid is prepared, according to 0.1-2ug/
The amount coating nature controlling line of cm；
S7, the assembling of seperation film and test strips, the assembling of test strips, it is characterised in that assemble the sample pad that secondary antibody is marked in S3
It to viscose carrier plastics plate one end, firmly presses, the film for being coated with nature controlling line and detection line is assembled in the middle part of test strips,
The other end of test strips pastes absorbing membrane, maintains sample lateral chromatography, test strips is put into plastic casing, by one section of sample pad
Plastic box bottom is placed on, plastic casing top is fixed in blotting paper one end；
S8, clinical sample detection, it is characterised in that according to heparin-binding protein standard curve, at room temperature in the sample pad of test strips
Position sequentially adds detection buffer solution and clinical samples, quantitatively detects heparin binding protein white level；
The heparin-binding protein calibration curve range in step sl, judges different infection indexs, it is characterised in that tight
HBP concentration in weight septic patient sample is preferably greater than 20ng/ml, dense for the HBP in meningitis clinical samples
Degree is preferably greater than 50ng/ml, is preferably greater than 50ng/ml for HBP concentration in the clinical samples of urinary tract infections.
The clinical samples need including but not limited to blood and urine according to infection detection in step sl,
The buffer solution added in step s 2 includes but not limited to PBS, HEPES, Tris buffer solution；The glucide of addition includes
But it is not limited to carbohydrates, the glucide additional proportions such as addition sucrose, trehalose, glucose and includes but not limited to 1%-10%；Add
The seralbumin entered includes but not limited to BSA, HSA, standard serum, and ratio includes but not limited to 1%-10%；.
2. a kind of immune microsphere chromatography detection heparin-binding protein Test paper preparation method according to claim 1,
It is characterized in that, microballoon or colour developing grain diameter include but not limited to 0.1-1 microns.
3. microballoon according to claim 2 or colour developing particle, it is characterised in that microballoon or colour developing particle include but not limited to
Fluorescent microsphere, color micro-sphere, preferably fluorescent microsphere.
4. sample pad according to claim 1 is coated with solid phase, it is characterised in that sample pad is coated with the secondary antibody of microballoon label,
The label secondary antibody can combine heparin-binding protein antibody and reacting pad nature controlling line coated antibody in detection buffer solution simultaneously.
5. reaction pad film according to claim 4 includes but not limited to nitrocellulose filter, cellulose acetate film.
6. a kind of immune microsphere chromatography detection heparin-binding protein Test paper according to claim 1, test paper inspection
The immune quantitative analyzer of Le Pu companies need to be inserted by surveying result, read the fluorescent value or OD value of sample to be tested.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710000257.0A CN108267592A (en) | 2017-01-03 | 2017-01-03 | A kind of immune microsphere chromatography detects heparin-binding protein Test paper |
Applications Claiming Priority (1)
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| CN109324184A (en) * | 2018-12-05 | 2019-02-12 | 厦门同仁心生物技术有限公司 | A kind of C hepatitis virus antigen fluorescence immune chromatography detection kit and preparation method thereof |
| CN109358197A (en) * | 2018-11-29 | 2019-02-19 | 中国农业科学院油料作物研究所 | A kind of fluorescence detection device and fluorescence detection method based on bridging antibody coupling fluorescent microsphere |
| CN109541220A (en) * | 2018-10-09 | 2019-03-29 | 温州启星生物技术有限公司 | The preparation method and application of C reactive protein, Procalcitonin, heparin-binding protein and serum amyloid A protein 1 |
| CN109541235A (en) * | 2018-08-19 | 2019-03-29 | 重庆早柒天生物科技股份有限公司 | Heparin-binding protein and Procalcitonin two joint detection reagent box and preparation method thereof |
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| CN110903375A (en) * | 2019-10-12 | 2020-03-24 | 南京立顶生物科技有限公司 | Method for producing and purifying heparin binding protein |
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| CN111562363A (en) * | 2019-06-25 | 2020-08-21 | 山西康健恩生物科技有限公司 | Heparin binding protein detection kit and preparation method thereof |
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| CN109541220A (en) * | 2018-10-09 | 2019-03-29 | 温州启星生物技术有限公司 | The preparation method and application of C reactive protein, Procalcitonin, heparin-binding protein and serum amyloid A protein 1 |
| CN109358197A (en) * | 2018-11-29 | 2019-02-19 | 中国农业科学院油料作物研究所 | A kind of fluorescence detection device and fluorescence detection method based on bridging antibody coupling fluorescent microsphere |
| CN109324184A (en) * | 2018-12-05 | 2019-02-12 | 厦门同仁心生物技术有限公司 | A kind of C hepatitis virus antigen fluorescence immune chromatography detection kit and preparation method thereof |
| CN111562363A (en) * | 2019-06-25 | 2020-08-21 | 山西康健恩生物科技有限公司 | Heparin binding protein detection kit and preparation method thereof |
| CN110824177B (en) * | 2019-09-11 | 2021-06-11 | 润和生物医药科技(汕头)有限公司 | Rapid detection test paper and preparation method and application thereof |
| CN110824177A (en) * | 2019-09-11 | 2020-02-21 | 润和生物医药科技(汕头)有限公司 | Rapid detection test paper and preparation method and application thereof |
| CN110903375A (en) * | 2019-10-12 | 2020-03-24 | 南京立顶生物科技有限公司 | Method for producing and purifying heparin binding protein |
| CN111208305A (en) * | 2020-02-16 | 2020-05-29 | 杨轶轩 | Application of HBP and PCT in preparation of septicemia diagnostic reagent |
| CN111208305B (en) * | 2020-02-16 | 2023-04-25 | 重庆翼锋生物科技有限公司 | Use of HBP and PCT in preparation of sepsis diagnostic reagent |
| CN111879938A (en) * | 2020-06-16 | 2020-11-03 | 烟台市疾病预防控制中心 | Quantum dot immunochromatography test strip for rapidly detecting pyrimethanil residues and preparation method |
| CN114324898A (en) * | 2022-03-11 | 2022-04-12 | 南京岚煜生物科技有限公司 | Binding pad treatment solution for heparin binding protein HBP detection |
| CN114324898B (en) * | 2022-03-11 | 2022-05-31 | 南京岚煜生物科技有限公司 | Binding pad treatment solution for heparin binding protein HBP detection |
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Application publication date: 20180710 |