CN109387640A - A kind of time-resolved fluoroimmunoassay chromatograph test strip and preparation method thereof that primary dcreening operation can be carried out to meningitis type - Google Patents
A kind of time-resolved fluoroimmunoassay chromatograph test strip and preparation method thereof that primary dcreening operation can be carried out to meningitis type Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
-
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention discloses a kind of time-resolved fluoroimmunoassay chromatograph test strips and preparation method thereof that primary dcreening operation can be carried out to meningitis type, can carry out the time-resolved fluoroimmunoassay chromatograph test strip of primary dcreening operation to meningitis type includes: sample bonding pad, detecting pad, sample suction pad and bottom plate, sample bonding pad, detecting pad and sample suction pad are successively set on bottom plate, and sample bonding pad and sample suction pad are laminated on respectively on the both ends of detecting pad, detecting pad is equipped with four detection lines and a nature controlling line.Sample size needed for the present invention is few, high sensitivity, and detection background signal is low, and high specificity, the range of linearity is wide, easy to operate, as a result reliably；Further, by the content for detecting PCT, CRP, ESAT in cerebrospinal fluid~6~CFP10 antigen and cryptococcus capsular polysaccharide antigen simultaneously, there can be a rough judgement to the type of meningitis, because a possibility that mistaken diagnosis occurs in empiricism when can effectively reduce clinical diagnosis, accurate immunology foundation is provided for making a definite diagnosis for meningitis type.
Description
Technical field
The present invention relates to a kind of time-resolved fluoroimmunoassay chromatograph test strip that can carry out primary dcreening operation to meningitis type and its
Preparation method belongs to field of biotechnology.
Background technique
Central nervous system infection is pediatric common disease and frequently-occurring disease, the most with meningitis and viral meningitis
It is common.In recent years, as the gene mutation of tubercle bacillus, antituberculotic development relatively lag behind and AIDS patient increases,
Domestic and international disease incidence lungy and case fatality rate gradually increase.Meanwhile it is wide with antibiotic, immunosuppressor and cortin
General application, body flora imbalance or immune function decline, so that the disease incidence of cryptococcal meningitis is also significantly raised.Due to this
The illness that several meningitis show has similitude, especially tubercular meningitis and cryptococcal meningitis clinically
Performance exactly likes, and is easy mistaken diagnosis, and sufferer timely cannot effectively treat to get killed.Therefore it is badly in need of finding a kind of energy early diagnosis
And identify the method for the meningitis cause of disease, to treatment, prognosis, lapses to, reduces patient and sequelae occur and there will be great clinical meaning.
It is numerous studies have shown that PCT's and CRP is horizontal aobvious in cerebrospinal fluid in bacillary and Patients with Virus Meningitis
It writes and is higher than healthy children, this illustrates that PCT and CRP can be used as children virus and meningitis clinical diagnosis in cerebrospinal fluid
Index.Since there is tuberculosis antigen in vivo first in infection mycobacterium tuberculosis (MTU), and tuberculosis antibody then to infect after 8 weeks
It rises and gradually generates, therefore detect the MTU secretion specific antigen in cerebrospinal fluid and can be used as quick diagnosis meninx active tuberculosis
Effective way.In infection early stage, pathological form mycobacterium tuberculosis can generally secrete a large amount of specific proteins, i.e., 6000~early stage
Exoantigen target (ESAT~6) and antigen culturing filtrate protein 10 (CFP10), and BCG vaccine is not present in both albumen
In BCG, therefore its marker that can be used as active tuberculosis.For cryptococcal meningitis, the country mostly use cerebrospinal fluid picture,
The methods of the dyeing of A Li Xinlan, ink dyeing and fungal culture, although fungal culture is to make a definite diagnosis the goldstandard of hidden brain, it is time-consuming compared with
It is long, 2~5d is needed, some fungies need 10d, and culture positive rate is low；Smear and ink dyeing positive rate are higher, especially to brain ridge
The case where microscopy after liquid centrifugation, can be improved positive rate, but still there are missing inspection and mistaken diagnosis, causes patient that cannot treat in time.
Summary of the invention
The present invention provide a kind of time-resolved fluoroimmunoassay chromatograph test strip that primary dcreening operation can be carried out to meningitis type and its
Preparation method, test strips of the present invention are used cooperatively fluorescence immunity analyzer by immunochromatographic method, can be convenient, is accurate and high
Detect that PCT, CRP, ESAT in cerebrospinal fluid~6~CFP10 antigen, cryptococcus capsular polysaccharide antigen contain simultaneously to sensitivity
Amount, for meningitis type it is first sentence certain theoretical foundation is provided.
In order to solve the above technical problems, the technical solution adopted in the present invention is as follows:
A kind of time-resolved fluoroimmunoassay chromatograph test strip that primary dcreening operation can be carried out to meningitis type, comprising: sample combines
Pad, detecting pad, sample suction pad and bottom plate, sample bonding pad, detecting pad and sample suction pad are successively set on bottom plate, and sample bonding pad
It is laminated on respectively with sample suction pad on the both ends of detecting pad, detecting pad is equipped with four detection lines and a nature controlling line.
The Testing index of above-mentioned test strips includes PCT, CRP, ESAT in cerebrospinal fluid~6~CFP10 antigen and cryptococcus pod
Film polysaccharide antigen；The antibody of sample bonding pad endoperidium having time resolved fluorometric microballoon label, time-resolved fluorescence microballoon label
Antibody be Testing index antibody.
In order to improve the accuracy of detection, the anti-PCT that time-resolved fluorescence microballoon label is coated on sample bonding pad is mono-
Clonal antibody, anti crp monoclonal antibody, anti-ESAT~6~CFP10 polyclonal antibody and anti-cryptococcus capsular polysaccharide monoclonal are anti-
Body.
In order to facilitate manufacture, and guarantee the accuracy of detection, time-resolved fluorescence microballoon is that the polystyrene of modified is micro-
Ball；Surfaces of Polystyrene Microparticles modifies one kind that functional group is carboxyl, hydroxyl or epoxy group, and the partial size of polystyrene microsphere is
100~500nm, preferably 180~300nm.
In order to improve the sensitivity of detection, the chelating of 1% (w/w) lanthanide series is filled with inside time-resolved fluorescence microballoon
Object, lanthanide series are one of europium, Shan, IGS or dysprosium.
Applicant it has been investigated that, using immunization measurement cerebrospinal fluid in cryptococcus capsular polysaccharide antigen content to hidden brain
Diagnosis and observation of curative effect have important clinical application value, high sensitivity is easy to operate, be suitble to promote；Time-resolved fluorescence is exempted from
It is a kind of non-isotop analysis technology that epidemic disease, which measures (TRFIA), it uses lanthanide series labelled antigen or antibody, according to group of the lanthanides member
The luminous characteristics of plain chelate measure fluorescence with TIME RESOLVED TECHNIQUE, while two parameters of Detection wavelength and time carry out signal
It differentiates, can effectively exclude the interference of non-specific fluorescence, greatly improve sensitivity for analysis, while there is marker preparation letter
Just, storage time length, "dead" pollution, detection is reproducible, easy to operate, standard curve range is wide, not by sample nature
The advantages that fluorescence interferes.
The method of above-mentioned time-resolved fluorescence microballoon label are as follows: including the following steps being connected in order:
1) time-resolved fluorescence microballoon is dissolved in buffer, the activation of activator isothermal vibration is added；
2) organic solvent will be added in the resulting material of step 1), after mixing, centrifugation, washing remove deactivator, with buffering
Microballoon is resuspended in liquid, and it is polyclonal to be then separately added into anti-PCT monoclonal antibody, anti crp monoclonal antibody, anti-ESAT~6~CFP10
Closing is added with buffer resuspension is redissolved after centrifugation in antibody and anti-cryptococcus capsular polysaccharide monoclonal antibody, isothermal vibration label
Agent is closed, and anti-PCT monoclonal antibody complex, the anti crp monoclonal antibody for obtaining time-resolved fluorescence microballoon label are multiple
Object, anti-ESAT~6~CFP10 Anti-TNF-α nanocrystal composition and anti-cryptococcus capsular polysaccharide monoclonal antibody complex are closed, is used for
The assessment of PCT, CRP, ESAT~6~CFP10 antigen and cryptococcus capsular polysaccharide antigen performance.
It is preferred that the activator in step 1) and step 2) is 1~(3~dimethylamino-propyl)~3~ethyl carbodiimide
Hydrochloride or/and N~HOSu NHS.
It is preferred that the method for time-resolved fluorescence microballoon label are as follows: including the following steps being connected in order:
1) activator EDC is added with being resuspended after MES buffer solution for cleaning 2 times of pH6100mM in time-resolved fluorescence microballoon
(1~(3~dimethylamino-propyl)~3~ethyl-carbodiimide hydrochloride) and NHS (N~HOSu NHS), makes them
Concentration be respectively 0.05%~0.1% and 0.1%~02%, 30 ± 5 DEG C of oscillations activate 60 ± 5min；
2) ethyl alcohol that volumetric concentration is 10% will be added in the resulting material of step 1), after mixing, centrifugation, washing are removed
EDC or NHS, be added buffer be resuspended microballoon, be separately added into again after mixing anti-PCT monoclonal antibody, anti crp monoclonal antibody,
Anti- ESAT~6~CFP10 polyclonal antibody and anti-cryptococcus capsular polysaccharide monoclonal antibody, 37 DEG C of oscillations mark 2h, after centrifugation
It is resuspended with buffer is redissolved, 10%BSA is added and closes 30min, the anti-PCT monoclonal for obtaining time-resolved fluorescence microballoon label is anti-
Nanocrystal composition, anti crp monoclonal antibody complex, anti-ESAT~6~CFP10 Anti-TNF-α nanocrystal composition and anti-cryptococcus pod membrane
Monoclonal Antibody against Polysaccharides compound.
Above-mentioned redissolution buffer includes Tris~Hcl, BSA, T~20, trehalose and NaN3；Preferably, buffer is redissolved
For Tris~Hcl (pH7.5~8.5) of 10~50mM, contain 0.05~0.1%BSA, 0.05~0.1%T~20,3~5%
Trehalose and 0.05%NaN3, preceding percentage is mass percent.
In order to avoid influencing each other, four detection lines are parallel to each other, and four detection lines are respectively can be glimmering with time resolution
The pairing antibody that the antibody specificity of light microballoon label combines is formed by coating, i.e., detects each target using double antibody sandwich method
The content of object.
It is preferred that four detection lines and a nature controlling line are parallel to each other, and set gradually along the length direction of detecting pad, from
It is successively four detection lines and a nature controlling line on sample bonding pad to the direction of sample suction pad.Namely four detection lines are detecting
Tandem on pad can in any permutation, and end of the nature controlling line apart from sample suction pad is nearest.
In order to improve the sensitivity and accuracy of detection, it is preferable that nature controlling line is coated with sheep anti-mouse igg, goat-anti chicken IgY or sheep
Anti-rabbit IgG.
As another preferred embodiment of the application, detecting pad includes 2~4 detection point pads, and four detection lines are randomly
It is distributed on detection point pad, every detection, which divides on pad, is equipped with a nature controlling line, on same detection point pad, compared to detection line matter
It is nearest from the end of sample suction pad to control line.
In order to reduce cost, while guaranteeing the sensitivity and accuracy of detection, detecting pad is nitrocellulose filter and is hole
The perforated membrane of 5~12um of diameter；The material of sample bonding pad is glass fibre element film or non-woven fabrics；The material of sample suction pad is water suction filter
Paper.
The preparation method of the above-mentioned time-resolved fluoroimmunoassay chromatograph test strip that primary dcreening operation can be carried out to meningitis type, including
The following steps being connected in order:
(1) processing of detecting pad:
It will test pad to be attached on bottom plate, take the pH7.4 phosphate buffer of 10~50mM containing 1~5wt% sucrose will
PCT monoclonal antibody, CRP monoclonal antibody, ESAT~6~CFP10 polyclonal antibody and the cryptococcus pod membrane of another epitope
Monoclonal Antibody against Polysaccharides are diluted to 1 ± 0.02mg/ml respectively, are used to prepare four detection lines；With containing 1~5wt% sucrose
Sheep anti-mouse igg antibody is diluted to 1 ± 0.02mg/ml by the phosphate buffer of 10~50mM, is used to prepare nature controlling line；By 1 ±
0.02 μ l/cm draws liquid measure, draws film instrument by biodot and examines uniform must draw to preparation on detecting pad of the antibody after above-mentioned 5 kinds dilutions
Survey line and nature controlling line；The detecting pad pulled is placed in 37 ± 3 DEG C of drying boxes, dry 3 ± 0.2h；
(2) processing of sample bonding pad:
After sample bonding pad carries out closing in advance with the buffer immersion containing surfactant, 37 ± 3 DEG C of dry 3h ±
0.2h；By the airjet spray head of Biodot instrument, the antibody of time-resolved fluorescence microballoon will be marked with according to 5 ± 0.02 μ l/
On the amount ullrasonic spraying to sample bonding pad of cm, sample bonding pad is prepared in 37 ± 3 DEG C of dry 3h ± 0.2h；
(3) it assembles:
By fixed one end for being laminated on the detecting pad that step (1) obtains of sample bonding pad that step (2) obtains, and sample will be inhaled
The fixed other end for being laminated on detecting pad of pad, laminating length is 1~4mm, is carried out with film instrument is cut out by the width of every 4~6mm
It cuts to get the time-resolved fluoroimmunoassay chromatograph test strip of primary dcreening operation can be carried out to meningitis type.
It is preferred that the buffer in step (2) is 100mM PB (pH7.4), wherein contain 2%NaCl, 2%BSA, 0.5%
Casein, 0.1%T~20 and 5% sucrose, preceding percentage are mass percent.
The unmentioned technology of the present invention is referring to the prior art.
The invention has the following advantages over the prior art: sample size needed for the present invention is few, high sensitivity detects background
Signal is low, and high specificity, the range of linearity is wide, easy to operate, as a result reliably；Further, by simultaneously detect PCT in cerebrospinal fluid,
The content of CRP, ESAT~6~CFP10 antigen and cryptococcus capsular polysaccharide antigen, can have to the type of meningitis one substantially
Judgement provide because a possibility that mistaken diagnosis occurs in empiricism when can effectively reduce clinical diagnosis for making a definite diagnosis for meningitis type
Accurate immunology foundation；Preparation method is simple, has promoted.
Detailed description of the invention
Fig. 1 is the knot for the time-resolved fluoroimmunoassay chromatograph test strip that one kind of the present invention can carry out primary dcreening operation to meningitis type
Structure schematic diagram.Wherein: 1 is sample bonding pad；2 be detecting pad；3 be sample suction pad；4 be bottom plate；T1, T2, T3 and T4 are respectively four
Detection line；C is nature controlling line.
Fig. 2 is that PCT log concentration value~fluorescence in the present invention counts logarithm standard curve.
Fig. 3 is that CRP log concentration value~fluorescence in the present invention counts logarithm standard curve.
Fig. 4 is that ESAT~6~CFP10 antigen concentration logarithm~fluorescence in the present invention counts logarithm standard curve.
Fig. 5 is that cryptococcus capsular polysaccharide antigen log concentration value~fluorescence in the present invention counts logarithm standard curve.
Specific embodiment
For a better understanding of the present invention, below with reference to the embodiment content that the present invention is furture elucidated, but it is of the invention
Content is not limited solely to the following examples.
Embodiment 1
As shown in Figure 1, one kind can carry out the time-resolved fluoroimmunoassay chromatograph test strip just sentenced to meningitis type, including
Bottom plate 4 is disposed with sample bonding pad 1, detecting pad 2 and sample suction pad 3, sample bonding pad 1 and sample suction pad 3 on the bottom plate 4
It is laminated on the both ends of detecting pad 3 respectively；The anti-PCT that time-resolved fluorescence microballoon label is coated on the sample bonding pad 1 is mono-
Clonal antibody, anti crp monoclonal antibody, anti-ESAT~6~CFP10 polyclonal antibody, anti-cryptococcus capsular polysaccharide monoclonal are anti-
Body；The detecting pad 2 is equipped with detection line T1, T2, T3, T4 and the nature controlling line C line being parallel to each other, T1, T2, T3, T4 and nature controlling line C
Line sets gradually and is parallel to each other along the length direction of detecting pad, and nature controlling line C line is nearest with a distance from sample suction pad 3, detection line
T1, T2, T3, T4 are coated with the monoclonal antibody for being surveyed another epitope of index, and nature controlling line C is coated with sheep anti-mouse igg.Detecting pad
For nitrocellulose filter and be 5~12um of aperture perforated membrane；The material of sample bonding pad is glass fibre element film；Sample suction pad
Material be absorbent filter.
Time-resolved fluorescence microballoon is the polystyrene microsphere of modified, and it is carboxylic that Surfaces of Polystyrene Microparticles, which modifies functional group,
Base, the partial size of polystyrene microsphere are 200nm；The chelate of 1% (w/w) europium of filling inside time-resolved fluorescence microballoon；Time
The method of resolved fluorometric microballoon label are as follows: by the 1mg time-resolved fluorescence microballoon MES buffer of the 100mM of 200 μ L pH=6
It is resuspended after cleaning 2 times, 1~(3~dimethylamino-propyl)~3~ethyl-carbodiimide hydrochloride and N~hydroxysuccinimidyl acyl is added
Imines (activator), making its concentration is respectively 0.08% and 0.15%, and 30 DEG C of oscillations activate 60min, and 10% ethyl alcohol 20ul is added,
Centrifuge washing removes deactivator after mixing, and microballoon is resuspended in the borate buffer that the 60mM of 200 μ L pH=7.5 is added again, mixes
It is separately added into the anti-PCT monoclonal antibody of 80ug, 80ug anti crp monoclonal antibody, anti-ESAT~6 80ug~more grams of CFP10 again afterwards
Grand antibody and the anti-cryptococcus capsular polysaccharide monoclonal antibody of 80ug, 37 DEG C of oscillations mark 2h, are resuspended after centrifugation with redissolution buffer,
10%BSA 50ul closing 30min (percentage is mass percent) is added, obtains the anti-PCT of time-resolved fluorescence microballoon label
Monoclonal antibody complex, anti crp monoclonal antibody complex, anti-ESAT~6~CFP10 Anti-TNF-α nanocrystal composition and resist hidden
Coccus capsular polysaccharide monoclonal antibody complex.Redissolution buffer is 0.03MTris~Hcl (pH7.5~8.5), includes
0.08%BSA, 0.08%T~20,4% trehalose and 0.05%NaN3, preceding percentage is mass percent.
Above-mentioned test strips can be prepared by following preparation method:
(1) nitrocellulose filter (NC film) is handled:
NC film is labelled to the designated position of bottom plate, take the pH7.4 phosphate buffer of the 50mM containing 1% sucrose by another
The PCT monoclonal antibody of epitope, CRP monoclonal antibody, ESAT~6~CFP10 polyclonal antibody, cryptococcus capsular polysaccharide Dan Ke
Grand antibody is diluted to 1mg/ml respectively, is used to prepare 4 T lines；With the phosphate buffer of the 50mM containing 1% sucrose by sheep anti mouse
IgG antibody is diluted to 1mg/ml, is used to prepare C line；Liquid measure is drawn by 1 μ l/cm, film instrument is drawn by biodot and dilutes above-mentioned 5 kinds
Antibody afterwards is uniform must to be drawn to preparation T line and C line on NC film；The NC film pulled is placed in 37 DEG C of drying boxes, dry 3h.
(2) processing of sample bonding pad:
Glass fibre element film with containing surfactant buffer (formula: 100mM PB (pH7.4), wherein containing 2%
NaCl, 2%BSA, 0.5% casein, 0.1%T~20 and 5% sucrose, preceding percentage are mass percent) impregnate progress
After pre- closing, 37 DEG C of dry 3h；By the airjet spray head of Biodot instrument, the antibody of time-resolved fluorescence microballoon will be marked with
According on the amount ullrasonic spraying to glass fibre element film of 5 μ l/cm, sample bonding pad is prepared in 37 DEG C of dry 3h.
(3) it assembles:
By fixed one end for being laminated on the nitrocellulose filter that step (1) obtains of sample bonding pad that step (2) obtains, and
By the fixed other end for being laminated on nitrocellulose filter of sample suction pad, the length that laminates at both ends is 2mm, presses every with film instrument is cut out
The width of 4mm is cut to get finished product.
The production of standard curve: firstly, the fluorescence of 6 various concentration standard items by kit operating procedure measurement PCT
It is worth (a:0ng/mL, b:0.5ng/mL, c:2ng/mL, d:10ng/mL, e:20ng/mL, f:40ng/mL), with standard concentration
Logarithm is abscissa, and the logarithm of fluorescent value is ordinate, is handled by double-log mathematical model Log~Log function, linear regression side
Journey is Y=0.8624X+3.0824, R2=0.9995.The TRFIA standard curve of PCT is shown in Fig. 2.
Similarly, by fluorescent value (a:1 μ g/ml, the b:5 μ of 8 various concentration standard items of kit operating procedure measurement CRP
G/ml, c:10 μ g/ml, d:50 μ g/ml, e:100 μ g/ml, f:150 μ g/ml, g:200 μ g/ml, h:250 μ g/ml), with standard
The logarithm of product concentration is abscissa, and the logarithm of fluorescent value is ordinate, is handled by double-log mathematical model Log~Log function, line
Property regression equation be Y=0.8851X+3.9226, R2=0.9994.The TRFIA standard curve of CRP is shown in Fig. 3.
Similarly, by the fluorescence of kit operating procedure measurement 5 various concentration standard items of ESAT~6~CFP10 antigen
It is worth (a:2.5ng/mL, b:25ng/mL, c:100ng/mL, d:500ng/mL, e:1600ng/mL), with the logarithm of standard concentration
For abscissa, the logarithm of fluorescent value is ordinate, is handled by double-log mathematical model Log~Log function, equation of linear regression is
Y=1.2223X+2.6403, R2=0.9999.ESAT~6~CFP10 antigen TRFIA standard curve is shown in Fig. 4.
Similarly, by the fluorescence of 5 various concentration standard items of kit operating procedure measurement cryptococcus capsular polysaccharide antigen
It is worth (a:1ng/mL, b:10ng/mL, c:50ng/mL, d:100ng/mL, e:500ng/mL), is cross with the logarithm of standard concentration
Coordinate, the logarithm of fluorescent value are ordinate, are handled by double-log mathematical model Log~Log function, equation of linear regression Y=
0.9689X+3.523, R2=0.9997.The TRFIA standard curve of cryptococcus capsular polysaccharide antigen is shown in Fig. 5.
Precision Experiment: high, normal, basic three standard concentrations of accurate quantification are measured using above-mentioned test strips, respectively
10 multiple holes are set.It the results are shown in Table 1.
1 Precision Experiment of table
As shown in Table 1: the variation within batch coefficient and interassay coefficient of variation of above-mentioned test strips≤10%, meet test paper rules and regulations
Provisioning request.
Accuracy experiment: recovery experiment is conventionally carried out.It the results are shown in Table 2.
2 recovery experiment of table
As shown in Table 2: the rate of recovery of low middle high three concentration of PCT is between 100.39~102.67%, average recovery rate
It is 101.19%；The rate of recovery of low middle high three concentration of CRP is between 99.09~100.20%, average recovery rate 99.6%；
Between 98.89~100.13%, average recovery rate is the rate of recovery of low middle high three concentration of ESAT~6~CFP10 antigen
99.63%；The rate of recovery of low middle high three concentration of cryptococcus capsular polysaccharide antigen is average to return between 99.45~100.20%
Yield is 99.87%.
Clinical application experiment:
186 meningitis children's positive cerebrospinal fluid of hospital are taken, wherein 58 are diagnosed as meningitis, 54 are diagnosed as
Viral meningitis, 46 are diagnosed as tubercular meningitis, and 28 are diagnosed as cryptococcal meningitis；114 healthy youngsters are taken again
Virgin feminine gender cerebrospinal fluid.
Specific detecting step: cerebrospinal fluid sample is added dropwise on sample bonding pad, and after standing 5~20min, test strips are inserted
Enter in fluorescence analyser matched with test strips, reads the fluorescence intensity level of C line, T1, T2, T3, T4 line respectively.
Meningitis Cerebrospinal Fluid in Patients PCT level is 12.94 ± 4.64ng/ml as the result is shown, and CRP level is
26.52±10.15μg/ml；Patients with Virus Meningitis cerebrospinal fluid PCT level is 0.98 ± 0.24ng/ml, and CRP level is
6.78±3.2μg/ml；Cerebrospinal Fluid of Patients with Tuberculous Meningitis ESAT~6~CFP10 antigen levels are 137 ± 48.4ng/ml；
Care of Patients with Cryptococcosis Meningitis cerebrospinal fluid cryptococcus capsular polysaccharide antigen level is 645 ± 234ng/ml；Healthy children cerebrospinal fluid
PCT level is 0.14 ± 0.11ng/ml, and CRP level is 1.01 ± 0.13 μ g/ml, and ESAT~6~CFP10 antigen levels are 2.4
± 1.38ng/ml, cryptococcus capsular polysaccharide antigen level is 1.14 ± 0.32ng/ml, compared to the hidden higher ESAT of brain patient
~6~CFP10 antigen and cryptococcus capsular polysaccharide antigen concentration, can almost ignore.Above-mentioned testing result meet it is bacillary,
PCT, CRP, ESAT~6~CFP10 antigen in viral, Tuberculous, Care of Patients with Cryptococcosis Meningitis and healthy children cerebrospinal fluid
And the reference value of cryptococcus capsular polysaccharide antigen, illustrate that this test strips detection sensitivity and accuracy are higher, it can be to meningitis class
Type carries out primary dcreening operation.
Embodiment 2
It is substantially the same manner as Example 1, except that: nature controlling line C is coated with goat-anti chicken IgY；Detecting pad includes 2 detections
Point pad, four detection lines are randomly distributed on 2 detection point pads, and every detection, which be divided on pad, two detection lines, and every is detected
Divide on pad and is equipped with a nature controlling line, it is nearest from the end of sample suction pad compared to detection line nature controlling line on same detection point pad.Face
Bed is consistent using result and embodiment 1, repeats no more.
Embodiment 3
It is substantially the same manner as Example 1, except that: nature controlling line C is coated with goat anti-rabbit igg；Detecting pad can also include
Four detection point pads, four detection lines are randomly distributed on four detection point pads, have a detection line on every detection point pad,
A nature controlling line is equipped on every detection point pad, same detection divides on pad, the end compared to detection line nature controlling line from sample suction pad
Portion is nearest.Clinical applications result and embodiment 1 are consistent, repeat no more.
Claims (10)
1. the time-resolved fluoroimmunoassay chromatograph test strip that one kind can carry out primary dcreening operation to meningitis type, it is characterised in that: include:
Sample bonding pad, detecting pad, sample suction pad and bottom plate, sample bonding pad, detecting pad and sample suction pad are successively set on bottom plate, and sample
Product bonding pad and sample suction pad are laminated on respectively on the both ends of detecting pad, and detecting pad is equipped with four detection lines and a nature controlling line.
2. the time-resolved fluoroimmunoassay chromatograph test strip of primary dcreening operation can be carried out to meningitis type as described in claim 1,
Be characterized in that: its Testing index includes PCT, CRP, ESAT-6-CFP10 antigen and cryptococcus capsular polysaccharide antigen in cerebrospinal fluid；
The antibody of sample bonding pad endoperidium having time resolved fluorometric microballoon label, the antibody of time-resolved fluorescence microballoon label are detection
The antibody of index.
3. the time-resolved fluoroimmunoassay chromatograph test strip of primary dcreening operation can be carried out to meningitis type as described in claim 1,
It is characterized in that: being coated with anti-PCT monoclonal antibody, the anti crp monoclonal of time-resolved fluorescence microballoon label on sample bonding pad
Antibody, anti-ESAT-6-CFP10 polyclonal antibody and anti-cryptococcus capsular polysaccharide monoclonal antibody.
4. the time-resolved fluoroimmunoassay chromatograph test strip of primary dcreening operation can be carried out to meningitis type as claimed in claim 2 or claim 3,
It is characterized by: time-resolved fluorescence microballoon is the polystyrene microsphere of modified；Surfaces of Polystyrene Microparticles modifies functional group
For one kind of carboxyl, hydroxyl or epoxy group, the partial size of polystyrene microsphere is 100~500nm.
5. the time-resolved fluoroimmunoassay chromatograph test strip of primary dcreening operation can be carried out to meningitis type as claimed in claim 2 or claim 3,
It is characterized by: being filled with the chelate of 1wt% lanthanide series inside time-resolved fluorescence microballoon, lanthanide series is europium, Shan, IGS
Or one of dysprosium.
6. the time-resolved fluoroimmunoassay chromatograph test strip of primary dcreening operation can be carried out to meningitis type as claimed in claim 2 or claim 3,
It is characterized by: the method for time-resolved fluorescence microballoon label are as follows: including the following steps being connected in order:
1) time-resolved fluorescence microballoon is dissolved in buffer, the activation of activator isothermal vibration is added；
2) organic solvent will be added in the resulting material of step 1), after mixing, centrifugation, washing remove deactivator, with buffer weight
Outstanding microballoon, be then separately added into anti-PCT monoclonal antibody, anti crp monoclonal antibody, anti-ESAT-6-CFP10 polyclonal antibody and
Anti- cryptococcus capsular polysaccharide monoclonal antibody, isothermal vibration label are added sealer and carry out with buffer resuspension is redissolved after centrifugation
Closing obtains the anti-PCT monoclonal antibody complex of time-resolved fluorescence microballoon label, anti crp monoclonal antibody complex, resists
ESAT-6-CFP10 Anti-TNF-α nanocrystal composition and anti-cryptococcus capsular polysaccharide monoclonal antibody complex, for PCT, CRP,
The assessment of ESAT-6-CFP10 antigen and cryptococcus capsular polysaccharide antigen performance.
7. the time-resolved fluoroimmunoassay chromatograph test strip of primary dcreening operation can be carried out to meningitis type as claimed in claim 6,
Be characterized in that: the activator in step 1) and step 2) be 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride or/
And n-hydroxysuccinimide；In step 2), organic solvent is the ethyl alcohol that volumetric concentration is 10%, redissolves buffer and includes
Tris-Hcl, BSA, T-20, trehalose and NaCl.
8. the time-resolved fluoroimmunoassay chromatograph test strip of primary dcreening operation can be carried out to meningitis type as claimed in claim 2 or claim 3,
It is characterized by: four detection lines are parallel to each other, four detection lines are respectively can be anti-with time-resolved fluorescence microballoon label
The pairing antibody of body specific binding is formed by coating；Four detection lines and a nature controlling line are parallel to each other, and along detection
The length direction of pad is set gradually, and is successively four detection lines and a Quality Control on the direction from sample bonding pad to sample suction pad
Line；Nature controlling line is coated with sheep anti-mouse igg, goat-anti chicken IgY or goat anti-rabbit igg.
9. the time-resolved fluoroimmunoassay as claimed in any one of claims 1-3 that can carry out primary dcreening operation to meningitis type chromatographs
Test strips, it is characterised in that: detecting pad includes 2~4 detection point pads, and four detection lines are randomly distributed on detection point pad,
A nature controlling line is equipped on every detection point pad, same detection divides on pad, and nature controlling line is closer from the end of sample suction pad than detection line；
Detecting pad be nitrocellulose filter and be 5~12um of aperture perforated membrane；The material of sample bonding pad be glass fibre element film or
Non-woven fabrics；The material of sample suction pad is absorbent filter.
10. the time-resolved fluoroimmunoassay described in any one of claim 1 to 9 that can carry out primary dcreening operation to meningitis type chromatographs
The preparation method of test strips, it is characterised in that: including the following steps being connected in order:
(1) processing of detecting pad:
It will test pad to be attached on bottom plate, take the pH7.4 phosphate buffer of 10~50mM containing 1~5wt% sucrose will be another
PCT monoclonal antibody, CRP monoclonal antibody, ESAT~6~CFP10 polyclonal antibody and the cryptococcus capsular polysaccharide of a epitope
Monoclonal antibody is diluted to 1 ± 0.02mg/ml respectively, is used to prepare four detection lines；With 10 containing 1~5wt% sucrose~
Sheep anti-mouse igg antibody is diluted to 1 ± 0.02mg/ml by the pH7.4 phosphate buffer of 50mM, is used to prepare nature controlling line；By 1 ±
0.02 μ l/cm draws liquid measure, draws film instrument by biodot and examines uniform must draw to preparation on detecting pad of the antibody after above-mentioned 5 kinds dilutions
Survey line and nature controlling line；The detecting pad pulled is placed in 37 ± 3 DEG C of drying boxes, dry 3 ± 0.2h；
(2) processing of sample bonding pad:
After sample bonding pad carries out closing in advance with the buffer immersion containing surfactant, 37 ± 3 DEG C of dry 3h ± 0.2h；It is logical
The airjet spray head for crossing Biodot instrument is super according to the amount of 5 ± 0.02 μ l/cm by the antibody for being marked with time-resolved fluorescence microballoon
Sound is sprayed on sample bonding pad, and sample bonding pad is prepared in 37 ± 3 DEG C of dry 3h ± 0.2h；
(3) it assembles:
By fixed one end for being laminated on the detecting pad that step (1) obtains of sample bonding pad that step (2) obtains, and sample suction pad is consolidated
Surely it is laminated on the other end of detecting pad, laminating length is 1~4mm, it is cut with film instrument is cut out by the width of every 4~6mm,
The time-resolved fluoroimmunoassay chromatograph test strip of primary dcreening operation can be carried out to meningitis type to obtain the final product.
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