CN103941019A - Immunofluorescence test paper strip for rapidly and quantitatively detecting L-FABP (Liver-Fatty Acid Binding Protein) and preparation method thereof - Google Patents
Immunofluorescence test paper strip for rapidly and quantitatively detecting L-FABP (Liver-Fatty Acid Binding Protein) and preparation method thereof Download PDFInfo
- Publication number
- CN103941019A CN103941019A CN201410141715.9A CN201410141715A CN103941019A CN 103941019 A CN103941019 A CN 103941019A CN 201410141715 A CN201410141715 A CN 201410141715A CN 103941019 A CN103941019 A CN 103941019A
- Authority
- CN
- China
- Prior art keywords
- pad
- fabp
- antibody
- conjugates
- test paper
- 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.)
- Pending
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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
-
- 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
Abstract
The invention discloses an immunofluorescence test paper strip for rapidly and quantitatively detecting L-FABP (Liver-Fatty Acid Binding Protein). The test paper strip comprises a test paper strip support piece 4 as well as a sample gasket 1, a detection membrane 2 and a water absorbing gasket 3 which are sequentially arranged on the test paper strip support piece 4 in a lap joint adhesion manner, wherein a coupler gasket 5 is arranged between the sample gasket 1 and the detection membrane 2; a first layer of glass fiber gasket 6-1 is arranged above one end of the coupler gasket 5, and a second layer of glass fiber gasket 6-2 is arranged below the other end of the coupler gasket 5, or only the first layer glass fiber gasket 6-1 is arranged above one end of the coupler gasket 5, or no gasket is arranged on the coupler gasket 5. The test paper strip is characterized in that a detection line 7 is arranged on the detection membrane 2; the detection line 7 is wrapped with an anti-L-FABP monoclonal antibody or polyclonal antibody; a control line 8 is arranged on the other side of the detection line 7 and is wrapped with an anti-streptavidin antibody; the coupler gasket 5 is coated with a fluorescence labeling L-FABP antibody coupler.
Description
Technical field
The invention belongs to clinical diagnose field, be specifically related to Immunofluorescence test paper strip of a kind of Quantitative detection L-FABP (liver type fatty acid binding protein) and preparation method thereof.
Background technology
L-FABP (liver type fatty acid binding protein) belongs to one of member of fatty acid binding protein (FABP) family, be one group low-molecular-weight can be in conjunction with the high conservative property cytoplasmic protein of long-chain fatty acid.Distribute according to its tissue specificity, 9 kinds of different FABP are isolated, be respectively liver type (L-FABP), little visible peristalsis visible intestinal peristalsis (I-FABP), myocardium type (H-FABP), adipocyte type (A-FABP), epidermis type (E-FABP), ileum type (II-FABP), brain type (B-FABP), myelin type (M-FABP), testis type (T-FABP), mainly in liver, small intestine and renal expression.In kidney, L-FABP mainly expresses at proximal convoluted tubule, there is the infringements such as High-grade Proteinuria, ischemic and toxicity during in kidney trouble; infer that L-FABP participates in the heavily absorption of urine free fatty acid at night; promote beta-oxidation energy supply, thereby alleviate oxidative stress damage, thus protection kidney.The people such as Yamamoto contact the perfusion of research renal ischemic/again and acute ischemic renal injury, send the not scope of nitre azoles display organization anoxia-induced apoptosis with anaerobic inaicator, found that, after ischemia/reperfusion when 2h, in the nephridial tissue of people L-FABP transgenic mice and wild mouse, all occur sending not nitre azoles positive region at kidney priopticon and cortex portion, but the damage range of transgenic mice is obviously less.Further under anaerobic condition, by detecting ROS level in transgenic mice promixal tubular cell and non-transfected cell, find that transgenic mice promixal tubular cell ROS level significantly declines compared with non-transfected cell, this result of study prompting high expressed kidney may play the effect of prevention promixal tubular cell generation ischemia/reperfusion injury.L-FABP molecular mass is less, only have 14.4KD, in the time that liver cell, renal tubular cell sustain damage, permeability of cell membrane changes can make it overflow fast, therefore L-FABP can be used as a sensitivity, special tissue damage mark, can well react the damage of renal tubule.
In kidney, the most noticeable clinical practice of L-FABP is as AKI(acute injury of kidney) diagnostic biomarkers.In the relation of a research urine L-FABP and AKI, 92 routine AKI patients and 62 examples are carried out to cross-sectional study without AKI patient, adopt ROC curve to evaluate the ability of L-FABP diagnosis AKI, ROC area under curve is 0.93, it is a good AKI diagnosis molecular marker, and research is pointed out, urinates L-FABP content higher, and patient's prognosis is poorer.
In addition, recent research shows, urine L-FABP can predict the generation of AKI well.The people's such as Portila research is found, 4h after CBP operation, and the content of the content of urine L-FABP in AKI patient is significantly higher than non-AKI patient's L-FABP content.According to ROC tracing analysis, AUC is 0.810, can be used as the biomarker of the rear independent prediction AKI risk of operation; In a clinical research, in infectious shock patient ICU, patient ICU of approximately 30% left and right, be detected and suffer from AKI, and result show L-FABP as a token of thing in the time judging the concurrent AKI of infectious shock patient, AUC on ROC curve is up to 0.99, in addition, result of study also shows, the content of urinating L-FABP in patient of dying of illness is significantly higher than the content of survivor's urine L-FABP, the content of urine L-FABP becomes conspicuousness positive correlation with mortality ratio, can illustrate that L-FABP is having extraordinary effect aspect prediction ICU patient's mortality ratio; And in the research of a contrast preparation and AKI relation, confirm in 66 patients, have 13 people before non-emergency angiography, just to occur that urine L-FABP significantly raises; Subsequently, there is the ephrosis that contrast preparation causes in 13 people, and urine L-FABP can be used as that prediction and diagnosis patient use contrast preparation and the contrast preparation acute injury of kidney (CI-AKI) that causes.
Along with other research is progressively goed deep into, urine L-FABP is presented at and detects the injury of kidney aspect that focal glomerulus necrosis, diabetic nephropathy, coronary contrast medium induced nephropathy, Cardiac bypass postoperative acute injury of kidney and kidney transplant ischemia-reperfusion cause, and has all shown extraordinary predicting function.So urine L-FABP can be used as the biomarker of extraordinary diagnosis AKI and prediction AKI.
Immunofluorescence technique (Fluorescence Immunoassay technique) is a kind of novel immunolabelling technique that is applied to antigen-antibody using fluorescence molecule as tracer label thing.Biotin-avidin system (Biotin-Avidin-System, BAS) is a kind of very effective biological respinse amplification system.Biotin-avidin system almost can be combined with the successful various labels of current research.Strong bonded and the multistage enlarge-effect of biotin-affinity element and labelled reagent high affinity, make that BAS is immune labeled and relevant tracer analysis is sensitiveer.It has become the new technology that is widely used at present trace antigen, antibody qualitative and quantitative analysis and position observation research.The huge superiority that BAS had in actual applications, is mainly manifested in the following aspects:
(1) biotin is easily combined with the biomacromolecule such as protein and nucleic acid, and the biotin derivative of formation has not only kept original biologically active of macromolecular substances, and higher than quiet degree, tool polyvalency.Therefore, BAS has multistage amplification, the sensitivity that makes it can greatly improve detection method in the time of application.
(2) combination between Avidin and biotin has high affinity, and its reaction is height selectivity.Therefore, the multi-level amplification of BAS, carrying the highly sensitive while, does not increase nonspecific interference.And BAS binding characteristic can be not influenced because of the high dilution of reaction reagent, make it can reduce to greatest extent in actual applications the non-specific effect of reaction reagent.
(3) Avidin can be 1,000,000 times of antigen-antibody reaction in conjunction with the affinity costant of biotin, and the two is very little in conjunction with the dissociation constant that forms compound, is non-reversible reaction; And acid, alkali, denaturant, protein resolvase and organic solvent all do not affect its combination.Therefore, in actual applications, the stability of product is high for BAS, improves the degree of accuracy of measuring.
(4) biotin and Avidin all can be made into multiple derivant, not only can be combined with all kinds of labelling techniques such as enzyme, fluorescein and radioactive nuclides, for detection of the Ag-Ab in body fluid, tissue or cell, hormone-acceptor and nucleic acid system and other various biological reaction systems, and can be made into affinity media, for separating of the reactant of purifying in above-mentioned each reaction system.
At present, the method that detects L-FABP is mainly Enzyme-multiplied immune technique (ELISA) at present, and elisa technique exists following shortcoming: checkout equipment requires high, and cost is high; Disturbing factor is more, and repeatability is bad; Detection time is long.Therefore Enzyme-multiplied immune technique detects L-FABP and is not suitable for clinical quick diagnosis.How can produce quantitative checkout equipment fast and become the problem that needs urgent solution.
Summary of the invention
An object of the present invention is to provide the Immunofluorescence test paper strip of a kind of Quantitative detection L-FABP, described Immunofluorescence test paper strip has the advantages such as convenient and swift, simple to operate, result is accurate, is suitable for clinical quick diagnosis.
Technical scheme of the present invention is:
The Immunofluorescence test paper strip of a kind of Quantitative detection L-FABP, it comprises the sample pad 1 that on test strips support chip 4 and test strips support chip 4, overlap joint is pasted in turn, detect film 2 and water suction pad 3, between described sample pad 1 and detection film 2, be provided with conjugates pad 5, conjugates pad 5 tops, one end are provided with ground floor glass fibre pad 6-1, its other end below is provided with second layer glass fibre pad 6-2 or conjugates pad 5 is only provided with ground floor glass fibre pad 6-1 or is not provided with arbitrary pad in top, one end, it is characterized in that: described detection film 2 is provided with detection line 7, described detection line 7 is coated with anti-L-FABP monoclonal antibody or polyclonal antibody, described detection line 7 another sides are provided with control line 8, control line 8 is coated with anti-Streptavidin (SAV) antibody, on described conjugates pad 5, be coated with fluorescence labeling L-FABP antibody conjugates.
Further, described Immunofluorescence test paper strip comprises a shell 10 outward, and described shell wraps up outside described colloid gold test paper, exposes detection line 7, control line 8 and adsorptive pads 3 on sample pad 1, nitrocellulose filter 4.
Further, in the pad of thing once in a while 5 of the present invention, the preparation of fluorescently-labeled L-FABP antibody conjugates comprises the steps:
(1) preparation of Biotin-L-FABP antibody
L-FABP antibody is dissolved in phosphate buffer (PBS), the biotin (Biotin) of activation is dissolved in its activation grade of guarantee in dimethyl sulfoxide (DMSO) (DMSO), then the L-FABP antibody being dissolved in phosphate buffer (PBS) is added in biotin (Biotin) solution of ready activation, stirring reaction, after having reacted, by solution ultrafiltration centrifugal concentrating, and repeatedly rinse, form Biotin-L-FABP antibody stoste, and calculate each L-FABP antibody may in conjunction with biotin (Biotin) mean number.
(2) preparation of fluorescence molecule-SAV
Anti-Streptavidin (SAV) powder is dissolved in activation in phosphate buffer (PBS), then add in fluorescence molecule powder and react being dissolved in anti-Streptavidin (SAV) in phosphate buffer (PBS), after having reacted, by solution ultrafiltration centrifugal concentrating, and repeatedly rinse, form fluorescence molecule-SAV stoste, and calculate each fluorescence molecule may in conjunction with the mean number of anti-Streptavidin (SAV).
(3) preparation of fluorescently-labeled L-FABP antibody conjugates
By the dilution of Biotin-L-FABP antibody stoste, add fluorescence molecule-SAV reaction according to the ratio of calculating, form fluorescently-labeled L-FABP antibody conjugates.
The preparation method that the Immunofluorescence test paper strip of a kind of Quantitative detection L-FABP is also provided when another object of the present invention, comprises the steps:
Step 1: prepare detection line; Detection film 2 for fixing coated antibody, is also immunoreactive nidus in Immunofluorescence test paper strip simultaneously; Detection line 7 is anti-L-FABP antibody to be used to the damping fluid dilutions such as 1*PBS, methyl alcohol, lines on described detection film 2, after then detecting film 2 and being fully dried and get final product;
Step 2: prepare control line; Described control line 8 is anti-Streptavidin (SAV) antibody to be used to the damping fluid dilutions such as 1*PBS, methyl alcohol, lines on described detection film 2, after then detecting film 2 and being fully dried and get final product;
Step 3: prepare conjugates pad; The starting material of described conjugates pad 5 are glass fiber filter, take out putting into for the preparation of the glass fiber filter of conjugates pad 5 after pre-sealing damping fluid soaks, after fully dry, fluorescently-labeled-L-FABP antibody conjugates is coated on conjugates pad 5, is fully drying to obtain with coating instrument;
Step 4: prepare sample pad; Described sample pad 1 can play preliminary filtering function by liquid towards sample, after sample pad is soaked with confining liquid, is fully drying to obtain;
Step 5: the assembling of test strips;
From bottom to top adhere to successively and detect film 2, water suction pad 3 and conjugates pad 5 at test strips support chip 4, on conjugates pad 5, be provided with ground floor glass fibre pad 6-1, second layer glass fibre pad 6-2 or only establish ground floor glass fibre pad 6-1 or be not provided with arbitrary pad, last layer is sample pad 1; By after above-mentioned material assembling, be cut into little, i.e. the Immunofluorescence test paper strip of the described quantitative detection L-FABP of system.
Or, also comprise a shell outward at described Immunofluorescence test paper strip, be prepared into the immunofluorescence paper box of quantitative detection L-FABP.Described shell wraps up outside described colloid gold test paper, exposes detection line 7, control line 8 and adsorptive pads 3 on sample pad 1, nitrocellulose filter 4.
The principle of work of the Immunofluorescence test paper strip of Quantitative detection L-FABP of the present invention is: adopt immune effluent reaction principle, be prepared from by double antibody sandwich method.L-FABP antigen when inspection in sample first respectively with coupling pad on L-FABP antibody conjugates generation immune response, form immune complex.Thereafter immune complex is along with sample chromatographic flow on NC Nitroncellulose film, when immune complex chromatography is during to detection zone (L-FABP detection line) on NC Nitroncellulose film, thus be coated in advance anti-L-FABP antibody on NC Nitroncellulose film and react and be fixed on the detection line of NC Nitroncellulose film.L-FABP in sample is more, and the compound on detection line is more, and the optical density value on band is just higher.Simultaneously, in testing process, unconjugated fluorescently-labeled L-FABP antibody conjugates also can detect chromatography on film with sample, when chromatography is when detecting the control line of film, thus fluorescently-labeled L-FABP can be coated in advance the anti-SAV antibody detecting on film and react and be fixed on control line.Fluorescence optical density value in sample in L-FABP concentration and detection line is proportional.
After reaction finishes, utilize detector that the optical density of control line and detection line is analyzed, and the analyze result obtaining is carried out to computing, thereby obtain relative optical density value (RI).Then detector calculates the concentration of L-FABP and shows result according to the typical curve having set in advance in detector, taking ng/mL as unit representation.
The Immunofluorescence test paper strip of Quantitative detection L-FABP of the present invention, has the advantages such as convenient and swift, simple to operate, result is accurate, is suitable for clinical quick diagnosis.
Brief description of the drawings
Fig. 1 is the structural representation of the Immunofluorescence test paper strip of Quantitative detection L-FABP of the present invention.
Wherein, 1: sample pad, 2: detect film, 3: water suction pad, 4: support chip, 5: conjugates pad, 6-1: ground floor glass fibre pad, 6-2: second layer glass fibre pad, 7: detection line, 8: control line.
Fig. 2 is the external structure schematic diagram of the immunofluorescence paper box of Quantitative detection L-FABP of the present invention.
Wherein, 10: shell, 1: sample pad, 7: detection line, 8: control line, 3: water suction pad.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in detail, can not be interpreted as it is limitation of the present invention;
The preparation of the Immunofluorescence test paper strip of [embodiment 1] a kind of Quantitative detection L-FABP
The Immunofluorescence test paper strip of a kind of Quantitative detection L-FABP as shown in Figure 1, it comprises the sample pad 1 that on test strips support chip 4 and test strips support chip, overlap joint is pasted in turn, detect film 2 and water suction pad 3, between described sample pad 1 and detection film 2, be provided with conjugates pad 5, conjugates pad 5 tops, one end are provided with ground floor glass fibre pad 6-1, its below, one end is provided with second layer glass fibre pad 6-2 or conjugates pad 5 is only provided with ground floor glass fibre pad 6-1 or is not provided with arbitrary pad in top, one end, described detection film 2 is provided with detection line 7, described detection line 7 is coated with anti-L-FABP monoclonal antibody or polyclonal antibody, described detection line 7 another sides are provided with control line 8, control line 8 is coated with anti-Streptavidin (SAV) antibody, on described conjugates pad 5, be coated with fluorescently-labeled L-FABP antibody conjugates.
Wherein, in the described pad of thing once in a while 5, the preparation of fluorescently-labeled L-FABP antibody conjugates comprises the steps:
(1) preparation of Biotin-L-FABP antibody:
L-FABP antibody is dissolved in phosphate buffer (PBS), the biotin (Biotin) of activation is dissolved in its activation grade of guarantee in dimethyl sulfoxide (DMSO) (DMSO), then the L-FABP antibody being dissolved in phosphate buffer (PBS) is added in biotin (Biotin) solution of ready activation, stirring reaction, after having reacted, by solution ultrafiltration centrifugal concentrating, and repeatedly rinse, form Biotin-L-FABP antibody stoste, and calculate each L-FABP antibody may in conjunction with biotin (Biotin) mean number;
(2) preparation of fluorescence molecule-SAV:
Anti-Streptavidin (SAV) powder is dissolved in activation in phosphate buffer (PBS), then add in fluorescence molecule powder and react being dissolved in anti-Streptavidin (SAV) in phosphate buffer (PBS), after having reacted, by solution ultrafiltration centrifugal concentrating, and repeatedly rinse, form fluorescence molecule-SAV stoste, and calculate each fluorescence molecule may in conjunction with the mean number of anti-Streptavidin (SAV);
(3) preparation of fluorescently-labeled L-FABP antibody conjugates:
By the dilution of Biotin-L-FABP antibody stoste, add fluorescence molecule-SAV reaction according to the ratio of calculating, form fluorescently-labeled L-FABP antibody conjugates;
Wherein, detection film 2 for fixing coated antibody, is also immunoreactive nidus in Immunofluorescence test paper strip simultaneously; Detection line 7 is anti-L-FABP antibody to be used to the damping fluid dilutions such as 1*PBS, methyl alcohol, lines on described detection film 2; Then will detect film 2 fully dry after and get final product;
Wherein, described control line 8 is anti-Streptavidin (SAV) antibody to be used to the damping fluid dilutions such as 1*PBS, methyl alcohol, lines on described detection film 2, after then detecting film 2 and being fully dried and get final product;
Wherein, prepare conjugates pad; The starting material of described conjugates pad 5 are glass fiber filter, take out putting into for the preparation of the glass fiber filter of conjugates pad 5 after pre-sealing damping fluid soaks, after fully dry, fluorescently-labeled L-FABP antibody conjugates is coated on conjugates pad 5 with coating instrument, is fully drying to obtain;
Prepare sample pad; Described sample pad 1 can play preliminary filtering function by liquid towards sample, after sample pad is soaked with confining liquid, is fully drying to obtain;
The assembling of test strips; Above-mentioned each building block is sticked in support pad 4 by structure shown in Fig. 1, obtain the Immunofluorescence test paper strip of Quantitative detection L-FABP.
[embodiment 2] is with Immunofluorescence test paper strip Quantitative detection L-FABP of the present invention
Get the Immunofluorescence test paper strip of Quantitative detection L-FABP of the present invention, specifically as shown in Figure 1, comprise the sample pad 1 that on test strips support chip 4 and test strips support chip 4, overlap joint is pasted in turn, detect film 2 and water suction pad 3, between described sample pad 1 and detection film 2, be provided with conjugates pad 5, conjugates pad 5 tops, one end are provided with ground floor glass fibre pad 6-1, its other end below is provided with second layer glass fibre pad 6-2 or conjugates pad 5 is only provided with ground floor glass fibre pad 6-1 or is not provided with arbitrary pad in top, one end, it is characterized in that: described detection film 2 is provided with detection line 7, described detection line 7 is coated with anti-L-FABP monoclonal antibody or polyclonal antibody, described detection line 7 another sides are provided with control line 8, control line 8 is coated with anti-Streptavidin (SAV) antibody, on described conjugates pad 5, be coated with fluorescence labeling L-FABP antibody conjugates.Detect according to the following steps: the urine specimen 1) gathering, preserve sample as low temperature and need return to room temperature.2) testing sample is added on sample pad 1 to reaction.3) interpretation, by described Immunofluorescence test paper strip be placed in (Rui Lai) immunofluorescence detector with and pertinent instruments result of determination.
[embodiment 3] is with immunofluorescence paper box Quantitative detection L-FABP of the present invention
Get the Immunofluorescence test paper strip of Quantitative detection L-FABP of the present invention, specifically as depicted in figs. 1 and 2, comprise the sample pad 1 that on test strips support chip 4 and test strips support chip 4, overlap joint is pasted in turn, detect film 2 and water suction pad 3, between described sample pad 1 and detection film 2, be provided with conjugates pad 5, conjugates pad 5 tops, one end are provided with ground floor glass fibre pad 6-1, its other end below is provided with second layer glass fibre pad 6-2 or conjugates pad 5 is only provided with ground floor glass fibre pad 6-1 or is not provided with arbitrary pad in top, one end, it is characterized in that: described detection film 2 is provided with detection line 7, described detection line 7 is coated with anti-L-FABP monoclonal antibody or polyclonal antibody, described detection line 7 another sides are provided with control line 8, control line 8 is coated with anti-Streptavidin (SAV) antibody, on described conjugates pad 5, be coated with fluorescence labeling L-FABP antibody conjugates.Wherein, described Immunofluorescence test paper strip comprises a shell 10 outward, and described shell wraps up outside described colloid gold test paper, exposes detection line 7, control line 8 and adsorptive pads 3 on sample pad 1, nitrocellulose filter 4.Detect according to the following steps: the urine specimen 1) gathering, preserve sample as low temperature and need return to room temperature.2) testing sample is added on sample pad 1 to reaction.3) interpretation, by described Immunofluorescence test paper strip be placed in (Rui Lai) immunofluorescence detector with and pertinent instruments result of determination.
Claims (4)
1. the Immunofluorescence test paper strip of a Quantitative detection L-FABP, it comprises the sample pad (1) that on test strips support chip (4) and test strips support chip (4), overlap joint is pasted in turn, detect film (2) and water suction pad (3), between described sample pad (1) and detection film (2), be provided with conjugates pad (5), top, conjugates pad (5) one end is provided with ground floor glass fibre pad (6-1), its below, one end is provided with second layer glass fibre pad (6-2) or conjugates pad (5) is only provided with ground floor glass fibre pad (6-1) or is not provided with arbitrary pad in top, one end, it is characterized in that: described detection film (2) is provided with detection line (7), described detection line (7) is coated with anti-L-FABP monoclonal antibody or polyclonal antibody, described detection line (7) another side is provided with control line (8), control line (8) is coated with anti-Streptavidin antibody, on described conjugates pad (5), be coated with fluorescence labeling L-FABP antibody conjugates.
2. the Immunofluorescence test paper strip of Quantitative detection L-FABP according to claim 1, it is characterized in that, described Immunofluorescence test paper strip comprises a shell (10) outward, described shell wraps up outside described colloid gold test paper, exposes detection line (7) and control line (8) and adsorptive pads (3) on sample pad (1), nitrocellulose filter (4).
3. the Immunofluorescence test paper strip of Quantitative detection L-FABP according to claim 1 and 2, is characterized in that: in the described pad of thing once in a while (5), the preparation of fluorescently-labeled L-FABP antibody conjugates comprises the steps:
(1) preparation of Biotin-L-FABP antibody:
L-FABP antibody is dissolved in phosphate buffer, the biotin of activation is dissolved in and in dimethyl sulfoxide (DMSO), ensures its activation grade, then the L-FABP antibody being dissolved in phosphate buffer is added in the biotin solution of ready activation, stirring reaction, after having reacted, by solution ultrafiltration centrifugal concentrating, and repeatedly rinse, form Biotin-L-FABP antibody stoste, and calculate each L-FABP antibody may in conjunction with biotin mean number;
(2) preparation of fluorescence molecule-SAV:
Anti-Streptavidin powder is dissolved in phosphate buffer and activates, then add in fluorescence molecule powder and react being dissolved in anti-Streptavidin in phosphate buffer, after having reacted, by solution ultrafiltration centrifugal concentrating, flushing, form fluorescence molecule-SAV stoste, calculate each fluorescence molecule may in conjunction with the mean number of anti-Streptavidin;
(3) preparation of fluorescently-labeled L-FABP antibody conjugates:
By the dilution of Biotin-L-FABP antibody stoste, add fluorescence molecule-SAV reaction according to the ratio of calculating, form fluorescently-labeled L-FABP antibody conjugates.
4. a preparation method for the Immunofluorescence test paper strip of Quantitative detection L-FABP, is characterized in that:
Comprise the steps:
Step 1: prepare detection line; Detection film (2) for fixing coated antibody, is also immunoreactive nidus in Immunofluorescence test paper strip simultaneously; Detection line (7) is anti-L-FABP antibody to be used to the damping fluid dilutions such as 1*PBS, methyl alcohol, lines described detection film (2) upper, then will detect film (2) fully dry, to obtain final product;
Step 2: prepare control line; Described control line (8) is anti-Streptavidin antibody to be used to the damping fluid dilutions such as 1*PBS, methyl alcohol, lines described detection film (2) upper, then will detect film (2) fully dry, to obtain final product;
Step 3: prepare conjugates pad; The starting material of described conjugates pad (5) are glass fiber filter, take out putting into for the preparation of the glass fiber filter of conjugates pad (5) after pre-sealing damping fluid soaks, after fully dry, with coating instrument, fluorescently-labeled-L-FABP antibody conjugates is coated on to conjugates pad (5) upper, is fully drying to obtain;
Step 4: prepare sample pad; Described sample pad (1) can play preliminary filtering function by liquid towards sample, after sample pad is soaked with confining liquid, is fully drying to obtain;
Step 5: the assembling of test strips;
From bottom to top adhere to successively and detect film (2), water suction pad (3) and conjugates pad (5) at test strips support chip (4), on conjugates pad (5), be provided with ground floor glass fibre pad (6-1), second layer glass fibre pad (6-2) or only establish ground floor glass fibre pad (6-1) or be not provided with arbitrary pad, last layer is sample pad (1); By after above-mentioned material assembling, be cut into little, i.e. the Immunofluorescence test paper strip of the described quantitative detection L-FABP of system.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410141715.9A CN103941019A (en) | 2014-04-10 | 2014-04-10 | Immunofluorescence test paper strip for rapidly and quantitatively detecting L-FABP (Liver-Fatty Acid Binding Protein) and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410141715.9A CN103941019A (en) | 2014-04-10 | 2014-04-10 | Immunofluorescence test paper strip for rapidly and quantitatively detecting L-FABP (Liver-Fatty Acid Binding Protein) and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103941019A true CN103941019A (en) | 2014-07-23 |
Family
ID=51188772
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410141715.9A Pending CN103941019A (en) | 2014-04-10 | 2014-04-10 | Immunofluorescence test paper strip for rapidly and quantitatively detecting L-FABP (Liver-Fatty Acid Binding Protein) and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103941019A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106443007A (en) * | 2016-08-31 | 2017-02-22 | 青岛汉唐生物科技有限公司 | Quantitative detection kit of serum amyloid protein A |
| CN107533053A (en) * | 2015-02-25 | 2018-01-02 | 积水医疗株式会社 | L FABP method of immunity and the measure reagent for methods described |
| CN116577498A (en) * | 2023-07-13 | 2023-08-11 | 济南玖方生物科技有限公司 | Application of sample pad for detecting HIV antibody in urine, test strip and sample pad treatment fluid |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102520194A (en) * | 2011-12-29 | 2012-06-27 | 深圳康美生物科技股份有限公司 | Fluorescence immunochromatographic assay method for quantitatively detecting heart fatty acid binding protein and kit for quantitatively detecting same |
| WO2013034264A1 (en) * | 2011-09-07 | 2013-03-14 | Roche Diagnostics Gmbh | L-fabp based diagnosis of kidney injury after an acute event or after a surgical intervention |
| CN103172941A (en) * | 2011-12-26 | 2013-06-26 | 苏州新波生物技术有限公司 | Polystyrene fluorescent nanometer particle as well as preparation method and applications thereof |
| CN103197063A (en) * | 2013-04-03 | 2013-07-10 | 瑞莱生物科技（江苏）有限公司 | Immunochromatography kit and detection method thereof |
-
2014
- 2014-04-10 CN CN201410141715.9A patent/CN103941019A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013034264A1 (en) * | 2011-09-07 | 2013-03-14 | Roche Diagnostics Gmbh | L-fabp based diagnosis of kidney injury after an acute event or after a surgical intervention |
| CN103172941A (en) * | 2011-12-26 | 2013-06-26 | 苏州新波生物技术有限公司 | Polystyrene fluorescent nanometer particle as well as preparation method and applications thereof |
| CN102520194A (en) * | 2011-12-29 | 2012-06-27 | 深圳康美生物科技股份有限公司 | Fluorescence immunochromatographic assay method for quantitatively detecting heart fatty acid binding protein and kit for quantitatively detecting same |
| CN103197063A (en) * | 2013-04-03 | 2013-07-10 | 瑞莱生物科技（江苏）有限公司 | Immunochromatography kit and detection method thereof |
Non-Patent Citations (3)
| Title |
|---|
| CANGEL P.Y. CHAN，ET AL: "Development of a quantitative lateral-flow assay for rapid detection of fatty acid-binding protein", 《JOURNAL OF IMMUNOLOGICAL METHODS》 * |
| TARO YONEKITA,ET AL: "Development of a novel multiplex lateral flow assay using an antimicrobial peptide for the detection of Shiga toxin-producing Escherichia coli", 《JOURNAL OF MICROBIOLOGICAL METHODS》 * |
| 宋巍 等: "肝脏型脂肪酸结合蛋白的重组表达及其单克隆抗体的制备和鉴定", 《中国科协第十四届年会第十七分会场—环境危害与健康防护研讨会论文集》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107533053A (en) * | 2015-02-25 | 2018-01-02 | 积水医疗株式会社 | L FABP method of immunity and the measure reagent for methods described |
| CN106443007A (en) * | 2016-08-31 | 2017-02-22 | 青岛汉唐生物科技有限公司 | Quantitative detection kit of serum amyloid protein A |
| CN116577498A (en) * | 2023-07-13 | 2023-08-11 | 济南玖方生物科技有限公司 | Application of sample pad for detecting HIV antibody in urine, test strip and sample pad treatment fluid |
| CN116577498B (en) * | 2023-07-13 | 2023-09-12 | 济南玖方生物科技有限公司 | Application of sample pad for detecting HIV antibody in urine, test strip and sample pad treatment fluid |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Gao et al. | Highly sensitive multianalyte immunochromatographic test strip for rapid chemiluminescent detection of ractopamine and salbutamol | |
| EP1618383B1 (en) | Membrane strip biosensor system for point-of-care testing | |
| CN103926401A (en) | Immunofluorescence test paper strip for rapidly and quantitatively measuring IGFBP-7 and TIMP-2 and preparation method thereof | |
| US20040018576A1 (en) | Bence Jones protein testing cassette | |
| JP5036867B2 (en) | Biosensor | |
| EP0981751B1 (en) | Immunoassay apparatus for diagnosis | |
| Nian et al. | Electrochemical immunoassay of cotinine in serum based on nanoparticle probe and immunochromatographic strip | |
| CN202149903U (en) | Time-resolved fluorescence immune chromatography quantitative detection test strip for C-reactive protein | |
| WO2008007359A2 (en) | Rapid diagnostic devices based on molecular imprinted polymers | |
| CN102087293A (en) | Immunochromatographic test strip for quantitatively detecting troponin I in whole course and preparation method thereof | |
| Cho et al. | Two-dimensional paper chromatography-based fluorescent immunosensor for detecting acute myocardial infarction markers | |
| CN101120253A (en) | Immunochromatographic test instrument and semiquantitative method using the same | |
| CN104865387A (en) | Immunofluorescence test strip for rapidly and quantitatively detecting ST2 (growth stimulation expressed gene 2) and NT-proBNP (N-terminal-pro-B-type-Natriuretic Peptide) and preparation method of immunofluorescence test strip | |
| CN103941007A (en) | Immunofluorescence test strip for fast and quantitatively detecting curative effect of aspirin and preparation method of immunofluorescence test strip | |
| CN105467116A (en) | Convenient procalcitonin detection kit | |
| CN103869079A (en) | Colloidal gold test paper for rapidly and quantificationally detecting galectin-3 | |
| CN106066399A (en) | The preparation method of the colloidal gold strip of detection early diabetic nephropathy | |
| KR101990301B1 (en) | Optical biosensor | |
| KR101916608B1 (en) | Biochemical-immunological hybrid biosensor and sensor system including the same | |
| CN105785041A (en) | Test strip for quantitatively detecting calprotectin, preparation method thereof and determining method for calprotectin concentration | |
| CN103941019A (en) | Immunofluorescence test paper strip for rapidly and quantitatively detecting L-FABP (Liver-Fatty Acid Binding Protein) and preparation method thereof | |
| CN203881772U (en) | Colloidal gold test paper for quickly and quantitatively detecting L-FABP | |
| KR20160120675A (en) | Rapid Quantitative Diagnostic Kit | |
| JPWO2003085402A1 (en) | Chromatographic test piece and method for producing the same | |
| Prado et al. | Electrochemical immunosensor for differential diagnostic of Wuchereria bancrofti using a synthetic peptide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20140723 |