CN107144693A - Detect CD4 in blood+Lateral chromatography kit of T cell quantity and preparation method thereof - Google Patents
Detect CD4 in blood+Lateral chromatography kit of T cell quantity and preparation method thereof Download PDFInfo
- Publication number
- CN107144693A CN107144693A CN201710301227.3A CN201710301227A CN107144693A CN 107144693 A CN107144693 A CN 107144693A CN 201710301227 A CN201710301227 A CN 201710301227A CN 107144693 A CN107144693 A CN 107144693A
- Authority
- CN
- China
- Prior art keywords
- chromatography
- layer
- sample pad
- sample
- hemofiltration
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- 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
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses CD4 in one kind detection blood+Lateral chromatography kit of T cell quantity and preparation method thereof.The kit includes sample processing tube and test strips；Sample processing tube contains the CD4 antibody of whole blood sample treatment fluid and signal substance markers；Test strips include bottom plate and are attached with bottom plate along the closely coupled sample pad in chromatography direction, hemofiltration layer, chromatography film layer and adsorptive pads；Sample pad part is pressed on hemofiltration layer, and hemofiltration layer segment is pressed in chromatography film layer, and water suction pad part is pressed in chromatography film layer；Detection line and nature controlling line are set in chromatography film layer, hemofiltration layer, detection line, nature controlling line and adsorptive pads are arranged in order.Immunocyte quantity in energy quick detection blood of human body of the invention is conducive to quickly making patient immune function evaluation, and the diagnosis and treatment for various diseases are all significant.
Description
Technical field
It is more particularly to a kind of to detect CD4 in blood the invention belongs to medical science detection technical field of immunoassay+T cell number
Lateral chromatography kit of amount and preparation method thereof.
Background technology
Find that between short more than 30 years, AIDS is in the whole world so far from the first in the world example patients infected hiv in 1981
Wreak havoc prevalence, it has also become great public health problem and social concern, cause the height of the World Health Organization and national governments
Pay attention to.
T lymphocytes are that a class has anti-infective and tumour immunity cell, and it is broadly divided into CD4+T cell and CD8+T is thin
Born of the same parents.CD4+T cell mainly plays assisting humoral immune and cellular immunity activation.CD4+The CD4 molecules on T cell surface are Chinese mugworts
The acceptor of disease virus is grown, after inhibition of HIV enters human body, it infects and damages CD4+T lymphocytes, cause humoral immunity of organism
It is suppressed with cellular immunity, causes whole immune function of human body to be destroyed, it is final to lose to the resistivities of various diseases
Cause death.Therefore CD4 can be passed through+The numeration of T lymphocytes can directly reflect HIV patients immune system's Injured status.
The CD4 of normal adult+T cell to contain 500~1600 in every microlitre of blood, patients infected hiv
CD4+T cell is it is possible that progressive or scrambling decline, and the immune system of prompting the infected receives serious infringement, when
CD4 in every microlitre of blood+When being less than 200 a variety of serious opportunistic infections or tumour may can occur for T cell.At present most
Newly provide the CD4 of HIV person in every microlitre of blood+T cell quantity levels should just proceed by treatment less than 350.
Check that the main method of AIDS includes at present：(1) body's immunity inspection, mainly including CD4+T cell number
Amount detection and CD4+T cell and CD8+T cell ratio is detected；(2) round pcr detection inhibition of HIV nucleic acid；(3) HIV antibody and anti-
Original detection, using enzyme linked immunosorbent assay, immunofluorescence assay, immune-blotting method method, lateral chromatography test paper method, gelatin
Grain agglutination test, radioimmunoprecipitation etc..CD4+The detection of T cell quantity is compared to viral nucleic acid detection and antiviral antibody inspection
Survey, can more intuitively reflect the immune state of patient, suitable therapic opportunity and therapeutic modality are provided for patient, so as to reach
To the purpose of accurate treatment, accurate medication, the treatment prevention and control for AIDS are significant.And currently for CD4+T is thin
Born of the same parents' quantity detection main method includes flow cytometry, micro-fluidic chip counting method and microscope people's work(counting method.Streaming
Cell art method and micro-fluidic chip counting method, with detection sample size is big and the advantage such as Detection accuracy height.But need costliness
The condition such as flow cytometer or micro-fluidic report control platform, the experiment technical personnel of specialty and specific experimental situation.These
Part is unfavorable for the real-time prevention and control of AIDS, is also unfavorable for the accurate diagnosis and treatment of AIDS in the general of the countries and regions of scarcity of resources
And.Microscope people's work(counting method, lower compared to Flow cytometry cost, instrument requirements are lower, are adapted to common test experience
Room, but it is big in test in laboratory, cumbersome, human error to need also exist for technical professional, be not suitable for quick detection and
Site Detection.
It is described in synthesis：The following Railway Project of method all generally existings on：First, it is necessary to the instrument and equipment of costliness；The
Two, it is necessary to testing laboratory and the technical professional of specialty；3rd, most of instrument cost and testing cost costliness is difficult to
Popularization.Lateral chromatography test strips have the following advantages that in the application of diagnostic field：First, without large-scale instrument and equipment；Second, behaviour
Make simple, can be used for Site Detection without professional and technical personnel；3rd, testing cost is low, and detection speed is fast, is adapted to promote and answers
With.But it is the egg in serum or blood plasma that lateral chromatography test paper on the market, which is used for the detection object of checkout and diagnosis field mainly, at present
White molecule or nucleic acid molecules, and for the detection of whole blood, lateral chromatography technology is detected also especially for cell quantity in blood
It is blank out.
The content of the invention
The primary and foremost purpose of the present invention is to overcome the shortcoming and deficiency of prior art to detect CD4 in blood there is provided one kind+T
The lateral chromatography kit of cell quantity.
Another object of the present invention is to provide the preparation method of above-mentioned lateral chromatography kit.
The purpose of the present invention is achieved through the following technical solutions：CD4 in one kind detection blood+The lateral layer of T cell quantity
Kit is analysed, sample processing tube and test strips is included；Wherein, sample processing tube contains whole blood sample treatment fluid and signal substance markers
CD4 antibody (beacon CD4 antibody)；Test strips include bottom plate and are attached with bottom plate along the closely coupled sample in chromatography direction
Pad, hemofiltration layer, chromatography film layer and adsorptive pads；Sample pad part is pressed on hemofiltration layer, and hemofiltration layer segment is pressed in chromatography film layer, is inhaled
Water cushion part is pressed in chromatography film layer；Detection line (T lines) and nature controlling line (C lines), hemofiltration layer, detection are set in chromatography film layer
Line, nature controlling line and adsorptive pads are arranged in order.
CD4 in described inspection detection blood+The lateral chromatography kit of T cell quantity, in addition to shell, shell are arranged at
The outside of test strips；Well and peep hole are provided with shell, well is located in sample pad, and peep hole is located at chromatographic film
Detection line and nature controlling line region on layer.The effect of plastic casing is to prevent test strips to be contaminated and portable easy-to-use.
The composition of described whole blood sample treatment fluid is as follows：0.9% is added in 0.02M, pH=7.3~7.4PB buffer solutions
(w/v) NaCl, 1% (w/v) BSA, 1% (w/v) PEG-4000,20% (v/v) saturated ammonium sulfate (22 ± 3 DEG C), 0.1% (w/
V) liquaemin, 0.02% (w/v) NaN3。
Amount of the described whole blood sample treatment fluid in the sample processing tube described in every is preferably 50 μ L.
Amount of the CD4 antibody of described signal substance markers in the sample processing tube described in every is preferably 1 μ L anti-by CD4
Body is calculated as the CD4 antibody of 0.01mg/mL signal substance markers.
The CD4 antibody of described signal substance markers is preferably the CD4 antibody of fluorescent microsphere mark.
The CD4 antibody of described fluorescent microsphere mark, is prepared preferably by following steps：In 100 μ L1% (w/
V) 400 μ L distilled waters are added in the fluorescent microsphere solution of carboxylated, then add 20 μ L 1 ‰ (w/v) EDC (carbodiimide) and
10 μ L 1 ‰ (w/v) NHS (n-hydroxysuccinimide), hybrid reaction 30min, the fluorescent microsphere solution after being activated；
The CD4 antibody-solutions that 60 μ L concentration are 1mg/mL are added after activation in fluorescent microsphere solution, are well mixed, under conditions of stirring
React 2h；It is subsequently added into the bovine serum albumin solution that 60 μ L concentration are 10% (w/v) to close, stirring reaction 1h；Centrifugation, is abandoned
Clearly, 60000 μ L are added and redissolve liquid resuspension precipitation, the CD4 antibody of fluorescent microsphere mark is obtained；Wherein, it is 0.02M, pH to redissolve liquid
5% (w/v) sucrose, 5% (w/v) trehalose, 0.5% (w/v) BSA, 0.5% (w/v) surface are added in=8.4 PB buffer solutions
Activating agent (S-17), 0.5% (v/v) Tween-20,0.5% (w/v) PEG-4000 and 0.01% (w/v) NaN3。
Described fluorescent microsphere solution is preferably that, to spend biological yellow-green fluorescence microspheres solution, fluorescent microsphere particle diameter is about
130nm, solid content are about 75 μm of ol/g, excitation waves for 1% (containing 10mg microsphere particles i.e. in 1mL solution), carboxyl-content
A length of 468nm, launch wavelength are 508nm.
The condition of described centrifugation is preferably 12000rpm centrifugations 15min.
The material of described bottom plate is impermeable water substance, preferably plastics, more preferably polyvinyl chloride (PVC).
The material of described hemofiltration layer is preferably V-9 type hemofiltration films.
Described hemofiltration layer is formed by stacking by least two layers of blood separating films；It is preferred that being formed by stacking by three metafiltration blood films；More preferably
The filter membrane that the filter membrane and length that the filter membrane for being 1.25cm for length, length are 1cm are 0.75cm is sequentially overlapped from top to bottom, wherein
Hemofiltration layer one end by chromatography film layer is smooth.The major function of hemofiltration layer is the haemocyte in filtering blood.Detection
Blood sample amount is generally 50-100 μ l, if using individual layer membrane filtration, the floor length of film length beyond test strips.
By experimental study, have been found that using three metafiltration blood films, straight length is also saved while experiment effect is reached.
Described chromatographic film is preferably nitrocellulose filter.
Sheep anti mouse secondary antibody layer is provided with described detection line (T).Sheep anti mouse secondary antibody is used for recognizing the mouse of signal substance markers
Source CD4+Antibody.Because the T4 antigen market price is expensive and unstable, if using it as T line envelope antigens, inspection will be caused
Survey cost too high, be not suitable for product promotion.Pass through experimental verification, CD4 of the sheep anti mouse secondary antibody to mouse source+Antibody has outstanding catch
Capacitation power, can meet requirement of experiment, and cost of manufacture have dropped nearly 100 times.
The rabbit igg layer of signal substance markers is provided with described nature controlling line (C).Nature controlling line mainly has two in the entire system
Individual effect：First as product quality control, directly judge whether product effective；Second, as than chromogenic indicator, can pass through T
The ratio reaction detection signal intensity of line and C lines, eliminates error interference.
The rabbit igg of described signal substance markers is preferably the rabbit igg of fluorescent microsphere mark.
The rabbit igg of described fluorescent microsphere mark, is prepared preferably by following steps：In 100 μ L 1% (w/v)
400 μ L distilled waters are added in the fluorescent microsphere solution of carboxylated, 20 μ L (w/v) EDC (carbodiimide) and 10 μ L (w/ are then added
V) NHS (n-hydroxysuccinimide), hybrid reaction 30min, the fluorescent microsphere solution after being activated；Fluorescence is micro- after activation
The rabbit igg solution that 30 μ L concentration are 2mg/mL is added in ball solution, is well mixed, 2h is reacted under conditions of stirring；Then plus
Enter the bovine serum albumin solution that 60 μ L concentration are 10% (w/v) to close, stirring reaction 1h；Centrifugation, abandons supernatant, adds 1000 μ L
Redissolve liquid and precipitation is resuspended, obtain the rabbit igg of fluorescent microsphere mark；Wherein, in redissolving liquid and being 0.02M, pH=8.4 PB buffer solutions
Add 5% (w/v) sucrose, 5% (w/v) trehalose, 0.5% (w/v) BSA, 0.5% (w/v) surfactant (S-17),
0.5% (v/v) Tween-20,0.5% (w/v) PEG-4000 and 0.01% (w/v) NaN3。
Described fluorescent microsphere solution is preferably that, to spend biological yellow-green fluorescence microspheres solution, fluorescent microsphere particle diameter is about
130nm, solid content are about 75 μm of ol/g, excitation wavelengths for 1% (containing 10mg microsphere particles in 1mL solution), carboxyl-content
It is 508nm for 468nm, launch wavelength.
The condition of described centrifugation is preferably 12000rpm centrifugations 15min.
The material of described sample pad is preferably glass fibre.
Described sample pad is to handle to obtain by whole blood sample pad treatment fluid, is comprised the following steps that：Every long × wide=
30cm × 2cm sample pad raw material add 5ml whole blood sample pad treatment fluids, and 37 DEG C are dried overnight, and humidity is less than 30%, normal temperature
Preserve；The composition of wherein whole blood sample pad treatment fluid is as follows：0.5% (w/ is added in pH value=7.4,0.015M PBSs
V) BSA, 2% (w/v) trehalose, 2% (w/v) PEG-4000,0.5% (v/v) Tween-20,0.1% (w/v) liquaemin,
0.02% (w/v) NaN3。
The material of described adsorptive pads is preferably thickening blotting paper.
CD4 in described detection blood+The preparation method of the lateral chromatography kit of T cell quantity, comprises the following steps：
(1) sample processing tube is prepared：
1. whole blood sample treatment fluid is prepared：100mg is added in 100mL 0.02M, the PB buffer solutions of pH value=7.3~7.4
Liquaemin, 0.9g NaCl, 1g BSA, 1g PEG-4000,20mL saturations (NH4)2SO4(22℃±3℃)、20mg NaN3；
2. a 200 μ L EP pipe (sterile), the CD4 added after 50 μ L whole blood samples treatment fluids and 1 μ L signal substance markers are taken
Antibody, obtains sample processing tube；The sample processing tube can be preserved 1 week at 23 DEG C ± 2 DEG C, and 4 DEG C can preserve 1 month, and -20 DEG C can protect
Deposit 3~6 months；
(2) test strips are prepared：
1. with whole blood sample pad treatment fluid processing sample pad, every long × wide=30cm × 2cm sample pad raw material add
Enter 5ml whole blood sample pad treatment fluids, 37 DEG C are dried overnight, humidity is preserved less than 30%, normal temperature；The group of whole blood sample pad treatment fluid
Into as follows：0.5% (w/v) BSA, 2% (w/v) trehalose, 2% (w/v) are added in pH value=7.4,0.015M PBSs
PEG-4000,0.5% (v/v) Tween-20,0.1% (w/v) liquaemin, 0.02% (w/v) NaN3；
2. the linear type of rabbit igg after sheep anti mouse secondary antibody and signal substance markers is coated in chromatographic film, forms inspection respectively
Survey line and nature controlling line；
4. first the chromatographic film that 2. step obtains is attached on bottom plate, obtains chromatographing film layer；Then close to chromatography film layer
Blotting paper is sticked in one end of nature controlling line, obtains adsorptive pads, the part of adsorptive pads is in chromatography film layer；Away from chromatography film layer Quality Control
Hemofiltration film is sticked in one end of line, obtains hemofiltration layer, and the part of hemofiltration layer is located in chromatography film layer；Sample pad, sample pad are sticked again
Part be located at hemofiltration layer on, obtain test strips；
(3) by sample processing tube together with test strips collocation, CD4 in detection blood is obtained+The lateral layer of T cell quantity
Analyse kit.
Sheep anti mouse secondary antibody described in step (2) preferably draws film instrument coating to chromatographic film by metal spraying, sheep anti mouse secondary antibody
Concentration be 1mg/mL when, by 1 μ l/cm prepare detection line.
The rabbit igg of signal substance markers described in step (2) preferably draws film instrument coating to chromatographic film by metal spraying, signal
The rabbit igg of substance markers prepares nature controlling line after diluting 14 times by 1 μ l/cm.
The present invention's is to be based on cells blocks competition law principle：Whole blood sample to be measured, sample are added into sample processing tube
Product dilute and pre-treatment through whole blood sample treatment fluid, the interference of the impurity such as reduction fibrin, while the letter in sample processing tube
Mark CD4 antibody is dyed to sample, forms beacon CD4 antibody and CD4+T cell compound；Then, by reacted mixing
Thing is added drop-wise in test strips, first, is handled by sample pad, and protein, buffer substance, sealer, hydrophilic is contained in sample pad
Property macromolecular substances, surfactant and anti-coagulants etc., these materials cause the permeability decrease of sample, and regulation pH value of solution is simultaneously
Immune response on NC films provides an optimal reaction environment；Secondly, whole cells in sample (including beacon CD4 antibody
And CD4+T cell compound) it can be blocked on hemofiltration layer, and other materials (including blood plasma, sample treatment liquid and remaining in sample
Under beacon CD4 antibody) pass through hemofiltration layer reach NC films；Sheep anti mouse secondary antibody capture beacon CD4 antibody formation detections on NC films
Signal.As CD4 in detection sample+T cell quantity is enough, can the beacon CD4 antibody in sample processing tube is most of or
When all combining, it is aobvious without color that the sheep anti mouse secondary antibody in detection line can not capture or can only capture on a small quantity beacon CD4 antibody
Show or color intensity is weaker, and the rabbit igg of the signal substance markers on nature controlling line, as fixed signal contrast line, detection line passes through
In contrast and interpretation cell quantity, is the moon in such case interpretation of the detection line color intensity less than nature controlling line color intensity
Property；As CD4 in blood+During T cell negligible amounts, it is not enough to be completely combined with beacon CD4 antibody in sample processing tube, so as to deposit
In beacon CD4 antibody, by hemofiltration layer, the sheep anti mouse secondary antibody on tested survey line is captured, and detection line has color to show, and color
Intensity is more than or equal to nature controlling line color intensity, and such case interpretation is the positive.
The present invention has the following advantages and effect relative to prior art：In energy quick detection blood of human body of the invention
Immunocyte quantity is conducive to quickly making patient immune function evaluation, all has weight for the diagnosis and treatment of various diseases
Big meaning.CD4 in the detection blood that the present invention is provided+The lateral chromatography kit of T cell quantity is traditional at present lateral
Design and improvement again has been carried out on chromatograph test strip architecture basics, and has added sample pre-treatments link, this technological break-through
Immuno-chromatographic assay technology extend to one by traditional immuno-chromatographic assay technology to detecting the limitation of sample object
Brand-new detection field, and in blood cell quantity detection field, promoted lateral chromatography technology develop, establish a kind of blood
The new method that cell quantity is detected in liquid.This technology requires low to raw material, it is only necessary to which a kind of single cell membrane surface is special
Antibody can just set up the quantity detection method of correspondence cell, the popularity with detection target.This technology is thin relative to traditional
Born of the same parents' detection technique has that simple to operate, instrument is portable, the low advantage of testing cost, and this invention will make up nowadays in the market and lack
Weary one kind can be carried out under amateur laboratory and amateur experimenter operation using portable set, energy simplicity,
The efficient immune state to AIDS patient is diagnosed and treated the product of prognosis.This technology sample processing time is
15min, detection time is 15min, can be examined according to the signal thing difference of CD4 antibody labelings using range estimation (collaurum) and instrument
(fluorescent microsphere, quantum dot) two kinds of result reading manners are surveyed, directly interpretation can go out yin and yang attribute result or by later data
Processing obtains specific cell quantity.
Brief description of the drawings
Fig. 1 is CD4 in the detection blood that the present invention is provided+The structural representation of the lateral chromatography kit of T cell quantity；
Wherein, 1- reaction tubes, 2- sample pads, 3- hemofiltrations layer, 4- chromatographies film layer, 5- detection lines, 6- nature controlling lines, 7- adsorptive pads, 8- bottom plates；
Arrow represents chromatographic solution flow direction.
Fig. 2 is the blood sample for the ELISA test strip dilution proportion that the present invention is provided, and passes through fluorescence intensity in detection line
The curve map obtained with the ratio and Sample Dilution ratio of fluorescence intensity on nature controlling line；Wherein, irregular curve obtains for detected value
The curve arrived, skew lines is matched curve.
Fig. 3 is the cd4 cell quantity gradient sample that the ELISA test strip passing ratio dilution that the present invention is provided is obtained, and is led to
Cross the curve map that the ratio and cell quantity of fluorescence intensity in fluorescence intensity and nature controlling line in detection line are obtained；Wherein, embed not
Regular curve figure is the curve that detection is obtained, and outside skew lines figure is with 50 cells/μ L~500 cell/μ L cell gradients
Fluorescence intensity the matched curve that is worth to of ratio.
Fig. 4 is whole blood sample pad prescription for the treatment of liquid optimum results figure of the present invention；Wherein figure a is the test knot of four kinds of sample pads
Fruit is schemed；It is heparin sodium content optimum results figure in the treatment fluid of whole blood sample pad 4 to scheme b；C is schemed in the treatment fluid of whole blood sample pad 4
PEG-400 content optimum results figures；It is BSA content optimum results figures in the treatment fluid of whole blood sample pad 4 to scheme d；Figure e is whole blood sample
Tween-20 contents optimum results figure in the treatment fluid of pad 4；It is content of trehalose optimum results in the treatment fluid of whole blood sample pad 4 to scheme f
Figure.
Embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited
In this.
Embodiment 1
Detect CD4 in blood+The preparation method and application of the lateral chromatography kit of T cell quantity：
1st, main agents material, as shown in table 1：
Table 1
2nd, instrument is mainly used, as shown in table 2：
Table 2
3rd, the preparation of main solution：
(1) 1% (m/v) citric acid three sodium solution：Weigh 1g trisodium citrates and be dissolved in 100mL ultra-pure waters；
(2) 1% (m/v) chlorauric acid solutions：1g gold chlorides are dissolved in 100mL ultra-pure waters；
(3)0.25mol/L K2CO3Solution：Weigh 3.45g K2CO3It is dissolved in 100mL ultra-pure waters；
(4) 1 ‰ (m/v) EDC solutions：Weigh 1mg EDC and be dissolved in 1mL ultra-pure waters；
(5)1‰(m/v)NHS：Weigh 1mg NHS and be dissolved in 1mL ultra-pure waters；
(6) 10% (m/v) BSA solution：10g BSA are weighed to be dissolved in 100mL ultra-pure waters；
(7) 0.15mol/L, PBS (10 ×), pH 7.4：NaCl 80g、KH2PO4 2g、KCl 2g、
Na2HPO4·12H2O 29g, add in 1000mL distilled waters and dissolve；
(8) 0.2mol/L, PB buffer solution (10 ×), pH 7.4：KH2PO4 4g、Na2HPO4·12H2O 29g, are added
Dissolved in 1000mL distilled waters, pH regulations to 7.4；
(9) 0.2mol/L, PB buffer solution (10 ×), pH 8.4：KH2PO4 4g、Na2HPO4·12H2O 29g, are added
Dissolved in 1000mL distilled waters, pH regulations to 8.4；
(10) liquid is redissolved：
It is added in 20mL 0.02mol/L pH=8.4 pB buffer solutions and dissolves；
(10) saturated ammonium sulfate：Ammonium sulfate 500g, with 60 DEG C of stirring and dissolvings of 500mL distilled waters water-bath, is cooled to room temperature, bottle
There is crystal precipitation at bottom.
(11) treatment fluid of whole blood sample pad 1：1g is added in 100mL pH=7.4,0.015M PBS (1 ×)
BSA, 1g trehalose, 1g PEG-4000,1mL Tween-20,20mg NaN3。
(12) treatment fluid of whole blood sample pad 2：0.8g is added in 100mL pH=7.4,0.015M PBS (1 ×)
NaCl, 1g acid hydrolyzed casein, 1g trehaloses, 1g PEG-4000,1g PVP-40K, 20mg NaN3。
(13) treatment fluid of whole blood sample pad 3：0.8g is added in 100mL pH=7.4,0.015M PBS (1 ×)
NaCl, 1g BSA, 1g trehalose, 1g PEG-4000,1mL Tween-20,100mg liquaemins, 20mg NaN3。
(14) treatment fluid of whole blood sample pad 4：1g is added in 100mL pH=7.4,0.015M PBS (1 ×)
BSA, 1g trehalose, 1g PEG-4000,1mL Tween-20,100mg liquaemins, 20mg NaN3。
(15) the whole blood sample pad treatment fluid after optimizing：0.5% (w/ is added in pH value=7.4,0.015M PBSs
V) BSA, 2% (w/v) trehalose, 2% (w/v) PEG-4000,0.5% (v/v) Tween-20,0.1% (w/v) liquaemin,
0.02% (w/v) NaN3。
(16) whole blood sample treatment fluid：0.9g NaCl, 1g are added in 100mL pH=7.4,0.02M PB buffer solutions
BSA, 1g PEG-4000,20mL saturated ammonium sulfate (22 ± 3 DEG C), 100mg liquaemins, 20mg NaN3。
4th, the preparation of kit
4.1 fluorescent microsphere
It is purchased from as the biological Co., Ltd of degree, particle diameter 130nm, solid content 1mg/mL, excitation wavelength 468nm, launch wavelength
508nm。
The preparation of the rabbit igg of 4.2 fluorescent microspheres mark
400 μ L ddH are added in the fluorescent microsphere solution of 100 μ L carboxylated2O, then adds 20 μ L (w/v) EDC and 10 μ L
(w/v) NHS, hybrid reaction 30min；Then 60 μ L rabbit iggs (1mg/mL) are added to be well mixed, are reacted under conditions of stirring
2h；Add the closing of the bovine serum albumin(BSA)s of 60 μ L 10%, stirring reaction 1h；Last 12000rpm centrifugations 15min abandons supernatant, adds
10000 μ L redissolve liquid and precipitation are resuspended, and respectively cleaning 6min with ultrasonic cleaning instrument high and low frequency makes solution fully be resuspended, and obtains glimmering
The rabbit igg (FNPs- rabbit iggs) of light microballoon mark, 4 DEG C of preservations.
The preparation of the CD4 antibody of 4.3 fluorescent microspheres mark
400 μ L ddH are added in the fluorescent microsphere solution of 100 μ L carboxylated2O, then adds 20 μ L (w/v) EDC and 10 μ L
(w/v) NHS, hybrid reaction 30min；Then 60 μ L CD4 antibody (1mg/ml) are added to be well mixed, it is anti-under conditions of stirring
Answer 2h；Add the closing of the bovine serum albumin(BSA)s of 60 μ L 10%, stirring reaction 1h；Last 12000rpm centrifugations 15min abandons supernatant, plus
Enter 10000 μ L and redissolve liquid resuspension precipitation, and respectively cleaning 6min with ultrasonic cleaning instrument high and low frequency makes solution fully be resuspended, and obtains
The CD4 antibody of fluorescent microsphere mark, 4 DEG C of preservations.
The processing of 4.4 sample pads
Sample pad processing：The whole blood sample that every long × wide=30cm × 2cm sample pad raw material are added after 5ml optimizations
Treatment fluid is padded, 37 DEG C are dried overnight, humidity is preserved less than 30%, normal temperature.
5th, the CD4 in the detection blood of human body based on fluorescent microsphere signal system+The immuno-chromatographic test paper strip of T cell quantity
Assembling
As shown in figure 1, the CD4 in the detection blood of human body based on fluorescent microsphere signal system+The lateral layer of T cell quantity
Analyse ELISA test strip system include reaction tube 1, sample pad 2, hemofiltration layer 3, chromatographic film 4, detection line 5, nature controlling line 6, adsorptive pads 7,
Bottom plate 8.
Reaction tube 1 adds 50 μ L whole blood sample treatment fluids, and 1 μ L fluorescent microspheres mark is then measured with micropipettor
CD4 antibody is added in reaction tube 1；Sheep anti-mouse igg (1mg/mL) and the FNPs- rabbit iggs of 14 times of dilution are drawn film instrument with metal spraying
With in 1 μ L/cm amount coating to chromatographic film (nitrocellulose filter) 4, as detection line 5 and nature controlling line 6, and in 37 DEG C of baking ovens
Dry 3h.Sample pad 2, hemofiltration layer 3, chromatographic film 4 and adsorptive pads 7 are sequentially mutually overlapped on bottom plate 8, obtained test paper plate is on request
Cut into the test strips of 3.51mm (width).Refill and get stuck that (plastics, which get stuck, is provided with well and detection hole, well into plastics
Position is located at sample pad location；The position of detection hole is located at detection line region) composition it is complete based on fluorescent microsphere signal
The CD4 of system+The immuno-chromatographic test paper strip of T cell quantity detection.
One better test strips, is that hemofiltration layer 3 is three layers, the filter that filter membrane that length is 1.25cm, length are 1cm
Film and length are sequentially overlapped from top to bottom for 0.75cm filter membrane, wherein being smooth by hemofiltration layer one end of chromatography film layer.
6th, cd4 cell quantity is detected based on fluorescent test paper strip
6.1 ELISA test strip flows
50 μ L anticoagulated whole bloods are taken to be added in sample processing tube, slight inhale plays mixing, reacts 15min；From sample processing tube
In take the 75 reacted liquid of μ L to be added to the wells of test strips, 15min fluorescence chart scanner interpretations.
The blood sample of 6.2 detection ratios dilution
By the whole blood sample of normal person carry out dilution proportion to different percentage concentrations (0%, 10%, 20%, 30%, 40%,
50%th, 60%, 70%, 80%, 90%, 100%), corresponding cell can also show different concentration gradients with this, then according to
Test paper testing process is operated.As a result it is as shown in Figure 2：By the ratio for calculating fluorescence intensity in fluorescence intensity and nature controlling line in detection line
Value, draws curve, and be fitted.This test paper rule can significantly distinguish the whole blood sample under different proportion dilution.
The diluted sample of the different cd4 cell concentration quantity of 6.3 detections
A：Absolute counting：1) the venous blood sample of normal person, is extracted with liquaemin vacuum anticoagulant tube；2), inhaled with pipettor
Take the 20 dyed blended liquid of μ L fluorescence antibodies (the streaming antibody staining liquid CD that streaming absolute counting is carried3CD4CD45Three color streamings resist
Body) it is added to above BD TrucountTMTubes, particle please don't be touched；3), 50 μ L anticoagulated whole bloods are drawn with pipettor to be added to
Above BD TrucountTMTubes, tube wall please don't be touched；4) counting lid, is covered, and lightly carries out vortex mixed,
15min is incubated under room temperature (20~30 DEG C) light protected environment；5) 450 μ L 1 × FACS hemolysins, are added into counting tube, are covered
Lid is counted, and lightly carries out vortex mixed, 15min is reacted in room temperature lucifuge；6), sample flow cytometer is divided
Analysis.
B：ELISA test strip：1), the data obtained by streaming carry out analysis calculating, and the cell number for obtaining sample is 689
Individual cell/μ L；2) it is, (0 cell/μ L, 50 cell/μ L, 100 cell/μ L, 200 thin according to cd4 cell quantity gradient
Born of the same parents/μ L, 300 cell/μ L, 400 cell/μ L, 500 cell/μ L, 600 cell/μ L) sample is diluted, prepare
Obtain the whole blood sample of different cd4 cell quantity；3), detected according to ELISA test strip flow.Testing result such as Fig. 3 institutes
Show：By calculating the ratio of fluorescence intensity in fluorescence intensity and nature controlling line in detection line, curve is drawn, and it is bent to obtain relevant criterion
Line, correlation well can be used for the quantitative analysis of cd4 cell quantity in whole blood.
7th, result is observed
Positive findings is detection line and fluorescence occurs in nature controlling line, and negative findings is that only nature controlling line fluorescence occurs；If Quality Control
Line does not have fluorescence, then the test strips are invalid.The sensitivity of colloidal gold strip is too low can not to meet detection requirement, fluorescent test paper strip
There is good correlation in the range of 50~500 cells/μ L, meet detection and require.
The design optimization of the hemofiltration film of embodiment 2
Pick several frequently seen hemofiltration film from the market first, carried out the evaluation of material cost performance, finally select
The V-9 type hemofiltration films of Hai Jiening bio tech ltd.By V-9 type hemofiltration films be cut into different length (0.5cm, 0.75cm,
1.0cm, 1.25cm, 1.5cm, 1.75cm, 2.0cm), test strips assembling is carried out by embodiment 1,75 μ L whole bloods are then added
This, is not in that blood is thin on NC films when hemofiltration film length reaches that the haemocyte in 75 μ L whole blood samples can be filtered clean by 2.0cm
The interference immune response of born of the same parents and signal occur.But the test strips total length in laboratory only has 6.0cm, long 1.5cm, NC film of adsorptive pads
Long 2.5cm, front end (sample pad, hemofiltration layer) only remain 2.0cm, and this test strips structure can not meet requirement.Therefore one is employed
Three layers of overlaying structure (0.75cm, 1.0cm, 1.25cm) of one end alignment are planted, the hemofiltration layer length after superposition is 1.25cm, structure
Meet and require, while the haemocyte in 75 μ L whole blood samples can be filtered into clean, occur without interference with immune response and signal
Embodiment 3 is optimized to sample pad and whole blood sample pad treatment fluid
(1) processing and assembling of sample pad
1. 4 kinds are selected to widely use to obtain blood testing sample pad prescription for the treatment of liquid configuration sample pad treatment fluid, with selecting glass
Glass tunica fibrosa (offer of Shanghai Jie Ning companies) is handled as sample pad former material with the treatment fluid of whole blood sample pad 1, whole blood sample pad 2
Liquid, the treatment fluid of whole blood sample pad 3 and the treatment fluid of whole blood sample pad 4 handle glass fibre membrane respectively, obtain 4 kinds of whole blood sample pads
(sample pad 1,2,3,4), while setting blank group, i.e., the glass fibre membrane handled without treatment fluid.
The step of whole blood sample pad treatment fluid handles glass fibre membrane is as follows：Glass fibre membrane according to it is every long × wide=
30cm × 2cm's cuts into blank sample pad, and every blank sample pad correspondence adds 5mL whole blood sample pad treatment fluids, and 37 DEG C are done
Dry to stay overnight, humidity is preserved less than 30%, normal temperature.
2. test strips are assembled into by embodiment 1.
3. 6.1 ELISA test strip flows in embodiment 1 are pressed, test strips test, the fluorescence that detection plasma sample is obtained is carried out
Intensity is most by force to be optimal.As a result as shown in fig. 4 a, the effect of the sample pad after 4 kinds of sample pad processing processing compares, sample pad 4
Effect is optimal, it is seen then that the effect of the treatment fluid of whole blood sample pad 4 is more excellent.Then each component of the treatment fluid of whole blood sample pad 4 is entered
One-step optimization.
(2) optimization of the treatment fluid of whole blood sample pad 4
The formula of the treatment fluid of whole blood sample pad 4 is optimized, first group be liquaemin concentration be respectively set to 0,
0.05%th, 0.1%, 0.15%, 0.2%, other compositions and concentration are with the treatment fluid of whole blood sample pad 4；Second group is PEG-4000
Concentration be respectively set to 0,0.5%, 1%, 1.5%, 2%, other compositions and concentration are with the treatment fluid of whole blood sample pad 4；3rd
Group is that BSA concentration is respectively set to 0,0.5%, 1%, 1.5%, 2%, and other compositions and concentration are handled with whole blood sample pad 4
Liquid；4th group is that Tween-20 concentration is respectively set to 0,0.5%, 1%, 1.5%, 2%, other compositions and the same whole blood of concentration
The treatment fluid of sample pad 4；5th group is that the concentration of trehalose is respectively set to 0,0.5%, 1%, 1.5%, 2%, other compositions and
Concentration is with the treatment fluid of whole blood sample pad 4.As a result as shown in Fig. 4 b~f, the whole blood sample pad treatment fluid after being optimized：
In 0.015M, pH=7.4 PBS add 0.5% (w/v) BSA, 2% (w/v) trehalose, 2% (w/v) PEG-4000,
0.5% (w/v) Tween-20,0.1% (w/v) liquaemin, 0.02% (w/v) NaN3。
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (10)
1. CD4 in one kind detection blood+The lateral chromatography kit of T cell quantity, it is characterised in that：Comprising sample processing tube and
Test strips；Wherein, sample processing tube contains the CD4 antibody of whole blood sample treatment fluid and signal substance markers；Test strips include bottom plate
Sample pad, hemofiltration layer, chromatography film layer and the adsorptive pads closely coupled with edge chromatography direction is attached with bottom plate；Sample pad part
It is pressed on hemofiltration layer, hemofiltration layer segment is pressed in chromatography film layer, water suction pad part is pressed in chromatography film layer；Set in chromatography film layer
Detection line and nature controlling line are put, hemofiltration layer, detection line, nature controlling line and adsorptive pads are arranged in order；
The composition of described whole blood sample treatment fluid is as follows：0.9% (w/v) is added in 0.02M, pH=7.3~7.4PB buffer solutions
NaCl, 1% (w/v) BSA, 1% (w/v) PEG-4000,20% (v/v) saturated ammonium sulfate, 0.1% (w/v) liquaemin, 0.02%
(w/v)NaN3；
Sheep anti mouse secondary antibody layer is provided with described detection line；
The rabbit igg layer of signal substance markers is provided with described nature controlling line；
Described sample pad is to handle to obtain by whole blood sample pad treatment fluid, is comprised the following steps that：Every long × wide=30cm
× 2cm sample pad raw material add 5ml whole blood sample pad treatment fluids, and 37 DEG C are dried overnight, and obtain sample pad；Wherein whole blood
The composition of product pad treatment fluid is as follows：0.5% (w/v) BSA, 2% (w/v) sea are added in pH value=7.4,0.015M PBSs
Algae sugar, 2% (w/v) PEG-4000,0.5% (w/v) Tween-20,0.1% (w/v) liquaemin, 0.02% (w/v) NaN3。
2. CD4 in inspection detection blood according to claim 1+The lateral chromatography kit of T cell quantity, it is characterised in that：
Also include shell, shell is arranged at the outside of test strips；Well and peep hole are provided with shell, well is located at sample
On pad, peep hole is located at detection line and nature controlling line region in chromatography film layer.
3. CD4 in inspection detection blood according to claim 1+The lateral chromatography kit of T cell quantity, it is characterised in that：
Amount of the described whole blood sample treatment fluid in the sample processing tube described in every is 50 μ L；
Amount of the CD4 antibody of described signal substance markers in the sample processing tube described in every is 1 μ L based on CD4 antibodies Antibodies
Calculate the CD4 antibody of the signal substance markers for 0.01mg/mL.
4. CD4 in inspection detection blood according to claim 1+The lateral chromatography kit of T cell quantity, it is characterised in that：
The CD4 antibody of described signal substance markers is the CD4 antibody that fluorescent microsphere is marked；
The rabbit igg of described signal substance markers is the rabbit igg that fluorescent microsphere is marked.
5. CD4 in inspection detection blood according to claim 4+The lateral chromatography kit of T cell quantity, it is characterised in that：
The CD4 antibody of described fluorescent microsphere mark, which is made by the steps, to be obtained：In 100 μ L 1% (w/v) carboxylated
400 μ L distilled waters are added in fluorescent microsphere solution, (w/v) EDC of 20 μ L 1 ‰ and 10 μ L 1 ‰ (w/v) NHS is then added, mixed
React 30min, the fluorescent microsphere solution after being activated；60 μ L concentration are added in fluorescent microsphere solution after activation for 1mg/mL
CD4 antibody-solutions, be well mixed, 2h is reacted under conditions of stirring；It is subsequently added into the ox blood that 60 μ L concentration are 10% (w/v)
Pure protein solution closing, stirring reaction 1h；Centrifugation, abandons supernatant, adds 60000 μ L and redissolves liquid resuspension precipitation, obtains fluorescence micro-
The CD4 antibody of ball mark；
The rabbit igg of described fluorescent microsphere mark, is made by the steps and obtains：In the glimmering of 100 μ L 1% (w/v) carboxylated
400 μ L distilled waters are added in light microspheres solution, 20 μ L (w/v) EDC and 10 μ L (w/v) NHS, hybrid reaction 30min are then added,
Fluorescent microsphere solution after being activated；The rabbit igg that 30 μ L concentration are 2mg/mL is added in fluorescent microsphere solution after activation molten
Liquid, is well mixed, 2h is reacted under conditions of stirring；It is subsequently added into the bovine serum albumin(BSA) that 60 μ L concentration are 10% (w/v) molten
Fluid-tight is closed, stirring reaction 1h；Centrifugation, abandons supernatant, adds 1000 μ L and redissolves liquid resuspension precipitation, obtains the rabbit of fluorescent microsphere mark
IgG；
Wherein, redissolve liquid be 0.02M, pH=8.4 PB buffer solutions in add 5% (w/v) sucrose, 5% (w/v) trehalose,
0.5% (w/v) BSA, 0.5% (w/v) surfactant S-17,0.5% (v/v) Tween-20,0.5% (w/v) PEG-4000
With 0.01% (w/v) NaN3。
6. CD4 in the inspection detection blood according to claim 4 or 5+The lateral chromatography kit of T cell quantity, its feature exists
In：
Described fluorescent microsphere solution has following features：Fluorescent microsphere particle diameter be about 130nm, solid content be 1%, carboxyl-content
About 75 μm ol/g, excitation wavelength are that 468nm, launch wavelength are 508nm.
7. CD4 in inspection detection blood according to claim 1+The lateral chromatography kit of T cell quantity, it is characterised in that：
The material of described bottom plate is impermeable water substance；
The material of described hemofiltration layer is V-9 type hemofiltration films；
Described chromatographic film is preferably nitrocellulose filter；
The material of described sample pad is glass fibre；
The material of described adsorptive pads is preferably thickening blotting paper.
8. CD4 in inspection detection blood according to claim 7+The lateral chromatography kit of T cell quantity, it is characterised in that：
Described hemofiltration layer is formed by stacking by least two layers of blood separating films.
9. CD4 in the detection blood described in any one of claim 1~8+The preparation side of the lateral chromatography kit of T cell quantity
Method, it is characterised in that comprise the following steps：
(1) sample processing tube is prepared：
1. whole blood sample treatment fluid is prepared：100mg heparin is added in 100mL 0.02M, the PB buffer solutions of pH value=7.3~7.4
Sodium, 0.9g NaCl, 1g BSA, 1g PEG-4000,20ml saturations (NH4)2SO4、20mg NaN3；
2. the CD4 antibody after the EP pipes that a 200 μ L are sterile, 50 μ L whole blood samples treatment fluids of addition and 1 μ L signal substance markers is taken,
Obtain sample processing tube；
(2) test strips are prepared：
1. with whole blood sample pad treatment fluid processing sample pad, every long × wide=30cm × 2cm sample pad raw material add 5ml
Whole blood sample pad treatment fluid, 37 DEG C are dried overnight；The composition of whole blood sample pad treatment fluid is as follows：100mL PH=7.4 0.015M
0.5g BSA, 2g trehalose, 2g PEG-4000,0.5mL Tween-20,100mg liquaemins, 20mg are added in PBS
NaN3；
2. the linear type of rabbit igg after sheep anti mouse secondary antibody and signal substance markers is coated in chromatographic film, forms detection line respectively
And nature controlling line；
4. first the chromatographic film that 2. step obtains is attached on bottom plate, obtains chromatographing film layer；Then close to chromatography film layer Quality Control
Blotting paper is sticked in one end of line, obtains adsorptive pads, the part of adsorptive pads is in chromatography film layer；Away from chromatography film layer nature controlling line
Hemofiltration film is sticked in one end, obtains hemofiltration layer, and the part of hemofiltration layer is located in chromatography film layer；Sample pad, the portion of sample pad are sticked again
Divide and be located on hemofiltration layer, obtain test strips；
(3) by sample processing tube together with test strips collocation, CD4 in detection blood is obtained+The lateral chromatography examination of T cell quantity
Agent box.
10. CD4 in inspection detection blood according to claim 1+The preparation method of the lateral chromatography kit of T cell quantity,
It is characterized in that：
Sheep anti mouse secondary antibody described in step (2) draws film instrument coating to chromatographic film by metal spraying, and the concentration of sheep anti mouse secondary antibody is
During 1mg/mL, detection line is prepared by 1 μ l/cm；
The rabbit igg of signal substance markers described in step (2) draws film instrument coating to chromatographic film by metal spraying, signal substance markers
Rabbit igg prepares nature controlling line after diluting 14 times by 1 μ l/cm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710301227.3A CN107144693B (en) | 2017-05-02 | 2017-05-02 | Detect the lateral chromatography kit and preparation method thereof of CD4+T cell quantity in blood |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710301227.3A CN107144693B (en) | 2017-05-02 | 2017-05-02 | Detect the lateral chromatography kit and preparation method thereof of CD4+T cell quantity in blood |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107144693A true CN107144693A (en) | 2017-09-08 |
| CN107144693B CN107144693B (en) | 2019-05-14 |
Family
ID=59775128
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710301227.3A Active CN107144693B (en) | 2017-05-02 | 2017-05-02 | Detect the lateral chromatography kit and preparation method thereof of CD4+T cell quantity in blood |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107144693B (en) |
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107703289A (en) * | 2017-10-30 | 2018-02-16 | 株洲市三三医院 | A kind of instant blood testing test strips and its paper box |
| CN108152500A (en) * | 2017-12-27 | 2018-06-12 | 江南大学 | A kind of Microcystin immune quantitative test paper item based on fluorescent microsphere label |
| CN108152515A (en) * | 2017-12-26 | 2018-06-12 | 公安部南昌警犬基地 | A kind of test paper detected for heat bitch progesterone and preparation method thereof |
| CN108732340A (en) * | 2018-06-04 | 2018-11-02 | 武汉纽康度生物科技股份有限公司 | A kind of chromatography buffer of removal red blood cell |
| CN108802392A (en) * | 2018-08-29 | 2018-11-13 | 上海市东方医院 | A kind of chromatograph test strip and its preparation method and application for diagnosis of prostate cancer |
| CN108918865A (en) * | 2018-05-21 | 2018-11-30 | 德康润生物科技（北京）有限公司 | Fluorescence immune chromatography test paper bar and reagent card |
| CN108982828A (en) * | 2018-06-28 | 2018-12-11 | 长春万成生物电子工程有限公司 | A kind of quantum dot examines the preparation and application of test paper fastly |
| CN110487704A (en) * | 2019-07-08 | 2019-11-22 | 暨南大学 | Immunofluorescence analysis device and immunofluorescence analysis method |
| CN111965349A (en) * | 2019-11-28 | 2020-11-20 | 上海荣盛生物药业有限公司 | Composition for fluorescence immunochromatography detection and application of composition in HIV detection |
| CN112763730A (en) * | 2020-12-29 | 2021-05-07 | 成都永安制药有限公司 | Colloidal gold immunochromatography trace whole blood detection test paper and detection method thereof |
| CN113015905A (en) * | 2018-11-19 | 2021-06-22 | 积水医疗株式会社 | Immunochromatography test strip and immunochromatography detection kit |
| CN113552338A (en) * | 2020-04-26 | 2021-10-26 | 格林生物医药科技(天津)有限公司 | Allergen specificity IgE antibody detection kit and preparation method thereof |
| CN114280312A (en) * | 2020-09-27 | 2022-04-05 | 河北特温特生物科技发展有限公司 | Whole blood separation membrane for immunofluorescence chromatography detection and preparation method and application thereof |
| CN116559429A (en) * | 2023-05-12 | 2023-08-08 | 广东省第二人民医院（广东省卫生应急医院） | Lateral chromatography kit for detecting HIV p24 antigen concentration and CD4+ T cell number |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090104630A1 (en) * | 2007-10-19 | 2009-04-23 | Reiter Paul C | Semi-quantitative immunochromatographic device and method for the determination of HIV/AIDS immune-status via measurement of soluble CD40 ligand/CD 154, A CD4+T cell equivalent |
| CN103323308A (en) * | 2013-05-23 | 2013-09-25 | 北京博晖创新光电技术股份有限公司 | Reagent for processing trace whole blood and application thereof |
| US8900850B2 (en) * | 2009-09-17 | 2014-12-02 | Michael J. Lane | Lateral flow based methods and assays for rapid and inexpensive diagnostic tests |
| CN104730231A (en) * | 2015-03-26 | 2015-06-24 | 北京乐普医疗科技有限责任公司 | Sample buffering solution used for fluorescence immunoassay quantitative determination and application of sample buffering solution |
| CN105067811A (en) * | 2015-07-22 | 2015-11-18 | 中国农业大学 | T-2 toxin detecting product based on fluorescent microsphere immunochromatography and preparation method thereof |
| CN205910194U (en) * | 2016-06-12 | 2017-01-25 | 吉林大学 | Synchronous immunity chromatography device of uniting different molecule form people neutrophil leucocyte lipid transporter of quantitative determination |
-
2017
- 2017-05-02 CN CN201710301227.3A patent/CN107144693B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090104630A1 (en) * | 2007-10-19 | 2009-04-23 | Reiter Paul C | Semi-quantitative immunochromatographic device and method for the determination of HIV/AIDS immune-status via measurement of soluble CD40 ligand/CD 154, A CD4+T cell equivalent |
| US8900850B2 (en) * | 2009-09-17 | 2014-12-02 | Michael J. Lane | Lateral flow based methods and assays for rapid and inexpensive diagnostic tests |
| CN103323308A (en) * | 2013-05-23 | 2013-09-25 | 北京博晖创新光电技术股份有限公司 | Reagent for processing trace whole blood and application thereof |
| CN104730231A (en) * | 2015-03-26 | 2015-06-24 | 北京乐普医疗科技有限责任公司 | Sample buffering solution used for fluorescence immunoassay quantitative determination and application of sample buffering solution |
| CN105067811A (en) * | 2015-07-22 | 2015-11-18 | 中国农业大学 | T-2 toxin detecting product based on fluorescent microsphere immunochromatography and preparation method thereof |
| CN205910194U (en) * | 2016-06-12 | 2017-01-25 | 吉林大学 | Synchronous immunity chromatography device of uniting different molecule form people neutrophil leucocyte lipid transporter of quantitative determination |
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107703289A (en) * | 2017-10-30 | 2018-02-16 | 株洲市三三医院 | A kind of instant blood testing test strips and its paper box |
| CN108152515A (en) * | 2017-12-26 | 2018-06-12 | 公安部南昌警犬基地 | A kind of test paper detected for heat bitch progesterone and preparation method thereof |
| CN108152500A (en) * | 2017-12-27 | 2018-06-12 | 江南大学 | A kind of Microcystin immune quantitative test paper item based on fluorescent microsphere label |
| CN108918865A (en) * | 2018-05-21 | 2018-11-30 | 德康润生物科技（北京）有限公司 | Fluorescence immune chromatography test paper bar and reagent card |
| CN108732340A (en) * | 2018-06-04 | 2018-11-02 | 武汉纽康度生物科技股份有限公司 | A kind of chromatography buffer of removal red blood cell |
| CN108982828A (en) * | 2018-06-28 | 2018-12-11 | 长春万成生物电子工程有限公司 | A kind of quantum dot examines the preparation and application of test paper fastly |
| CN108802392A (en) * | 2018-08-29 | 2018-11-13 | 上海市东方医院 | A kind of chromatograph test strip and its preparation method and application for diagnosis of prostate cancer |
| CN113015905A (en) * | 2018-11-19 | 2021-06-22 | 积水医疗株式会社 | Immunochromatography test strip and immunochromatography detection kit |
| CN110487704A (en) * | 2019-07-08 | 2019-11-22 | 暨南大学 | Immunofluorescence analysis device and immunofluorescence analysis method |
| CN111965349A (en) * | 2019-11-28 | 2020-11-20 | 上海荣盛生物药业有限公司 | Composition for fluorescence immunochromatography detection and application of composition in HIV detection |
| CN113552338A (en) * | 2020-04-26 | 2021-10-26 | 格林生物医药科技(天津)有限公司 | Allergen specificity IgE antibody detection kit and preparation method thereof |
| CN114280312A (en) * | 2020-09-27 | 2022-04-05 | 河北特温特生物科技发展有限公司 | Whole blood separation membrane for immunofluorescence chromatography detection and preparation method and application thereof |
| CN114280312B (en) * | 2020-09-27 | 2023-09-15 | 河北特温特生物科技发展有限公司 | Whole blood separation membrane for immunofluorescence chromatography detection and preparation method and application thereof |
| CN112763730A (en) * | 2020-12-29 | 2021-05-07 | 成都永安制药有限公司 | Colloidal gold immunochromatography trace whole blood detection test paper and detection method thereof |
| CN116559429A (en) * | 2023-05-12 | 2023-08-08 | 广东省第二人民医院（广东省卫生应急医院） | Lateral chromatography kit for detecting HIV p24 antigen concentration and CD4+ T cell number |
| CN116559429B (en) * | 2023-05-12 | 2024-01-23 | 广东省第二人民医院（广东省卫生应急医院） | Lateral chromatography kit for detecting HIV p24 antigen concentration and CD4+ T cell number |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107144693B (en) | 2019-05-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107144693B (en) | Detect the lateral chromatography kit and preparation method thereof of CD4+T cell quantity in blood | |
| KR102322094B1 (en) | Method and device for combined detection of viral and bacterial infections | |
| CN101939645B (en) | Cell assay kit and method | |
| US9523682B2 (en) | Methods and systems for detecting an analyte in a sample | |
| CN102072957B (en) | Hepatitis C virus antibody diagnostic kit and preparation method thereof | |
| CN101711361B (en) | The multiplex method that detection is infected | |
| EP3896447A1 (en) | A lateral flow detection device for detecting a coronavirus by immunoassay | |
| CN107941706A (en) | system and method for measuring chemical state | |
| CN107765002A (en) | A kind of colloidal gold immuno-chromatography test paper strip and its preparation method and application | |
| CN101387648A (en) | Erythrocyte membrane antigen magnetic ball kit and applications on blood group antibody detection | |
| CN101858914B (en) | Reagent strip for testing syphilis specific total antibodies through gold immunochromatographic assay and preparation method thereof | |
| CN104198716A (en) | Animal brucella antibody gold mark rapid detection kit | |
| CN105717308A (en) | Immunochromatography kit for fast and quantitatively detecting fecal lactoferrin | |
| CN101825635B (en) | Reagent strip for joint detection of syphilis specific IgG antibody and specific total antibody and preparation method thereof | |
| CN101825636A (en) | Reagent strip for joint detection of syphilis specific IgM antibody and specific total antibody and preparation method thereof | |
| CN109342736A (en) | A kind of microtrabeculae agglutination antihuman globulin detection card | |
| CN101858915A (en) | Reagent strip for testing syphilis specific IgG antibodies through gold immunochromatographic assay and preparation method thereof | |
| CN201673156U (en) | Colloidal gold immunochromatographic test reagent strip for syphilis specific IgM antibody | |
| CN105486871B (en) | A kind of quick detection canine parvovirus antibody blood clotting suppresses colloidal gold strip, kit and the detection method of potency | |
| CN110488012A (en) | A kind of auxiliary diagnostic box of pneumonia of newborn and application | |
| Darton et al. | Assessment and translation of the antibody-in-lymphocyte supernatant (ALS) assay to improve the diagnosis of enteric fever in two controlled human infection models and an endemic area of Nepal | |
| CN103421747A (en) | Hybridoma cell strain capable of secreting bovine IL-4 monoclonal antibody, monoclonal antibody secreted by hybridoma cell strain and application of hybridoma cell strain | |
| CN204177807U (en) | Helicobacter Pylori urease antibody IgM/IgG associating gold mark test card | |
| CN104090100A (en) | Brucella antibody gold-labeled fast detection kit | |
| CN209102728U (en) | A kind of microtrabeculae agglutination antihuman globulin detection card |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |