CN107271692A - Fluorescent microsphere for marking specific high-affinity recombinant antibody and application thereof - Google Patents
Fluorescent microsphere for marking specific high-affinity recombinant antibody and application thereof Download PDFInfo
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- CN107271692A CN107271692A CN201710691668.9A CN201710691668A CN107271692A CN 107271692 A CN107271692 A CN 107271692A CN 201710691668 A CN201710691668 A CN 201710691668A CN 107271692 A CN107271692 A CN 107271692A
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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
-
- 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
- G01N33/577—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
Abstract
The invention belongs to the field of biological medicine, and particularly relates to a fluorescent microsphere for marking a specific high-affinity recombinant antibody, a preparation method and application thereof, in particular to a plug-in fluorescent detection card for quickly and quantitatively detecting KIM-1, NGAL, IGFBP7, TIMP-2, IL-18, PCT and cTnI in a human urine sample or a blood sample. The method for marking the fluorescent microspheres of the specific high-affinity recombinant antibody specifically comprises the steps of firstly activating the fluorescent microspheres; then putting the activated fluorescent microspheres into a dialysis bag filled with a phosphate buffer solution to obtain a dialyzed fluorescent microsphere solution; adding amino polyethylene glycol carboxyl, further activating the fluorescent microsphere solution after secondary dialysis, marking by using a recombinant antibody, centrifuging after reaction, cleaning the precipitate, and then resuspending the precipitate. The fluorescent microsphere provided by the invention can obviously improve the reaction off-line of immunofluorescence, so that the sensitivity of the prepared detection card is improved by 10-100 times compared with that of the traditional method, and the detection range is also obviously improved.
Description
Technical field
The invention belongs to biomedicine field, the glimmering of specific high-affinity recombinant antibodies is marked in particular to a kind of
Kim1 in light microballoon and preparation method and application, particularly a kind of Quantitative detection people urine sample or blood sample
(KIM-1), the related apolipoprotein (NGAL) of neutrophil leucocyte gelatinase, insulin-like growth factor binding protein-7 (IGFBP-
7), tissue inhibitor of metalloproteinase -2 (TIMP-2), interleukin 18 (IL-18), Procalcitonin (PCT), cardiac muscle troponin I
(cTnI) i.e. slotting fluoroscopic examination card.
Background technology
The transmembrane glycoprotein that KIM-1 (Kim1) is made up of 334 amino acid residues, in normal kidney tissue
Hardly express, but be in high expression status in people's proximal tubular epithelial cells after ischemic and injury of kidney, its extracellular domain exists
Soluble fragments are degraded under metallo-matrix proteases effect and are discharged into extracellular, and are discharged into urine, as acute injury of kidney
One important Testing index.Relative to serum creatinine and some other mark, KIM-1 is no matter in terms of specificity or sensitiveness
There is its clear superiority, and it has higher specificity to distinguishing proximal tubule damage.
Clinically judge that the most frequently used biological indicator of people's injury of kidney is serum creatinine concentration at present, but serum creatinine only has
Can just detect concentration later to a certain degree in injury of kidney has significant change, and serum creatinine is not that the ideal early diagnosed refers to
Mark.Though the newer biological indicator serum bladder chalone C clinically occurred in recent years can more sensitive reflection injury of kidney degree, it is special
It is not to assess glomerular filtration function to be damaged the lesion for waiting correlation, but still early stage acute injury of kidney (AKI) can not be reacted.It is newest to grind
Hair finds that KIM-1 is not expressed as Novel marker in normal kidney tissue, on the proximal convoluted tubule after ischemic and injury of kidney
In high expression in skin, discovery kidney early lesion that can be promptly and accurately, for the monitoring of the AKI state of an illness, prognosis evaluation, accurate diagnosis and treatment
With very important value.
Due to KIM-1 contents KIM as little as pg/ml in urine, such a measurement mark is detected, in the urgent need to high accurate
Degree, the antibody of sensitivity and matched detection method.Simultaneously quick detection in AKI diagnosis relatively urgently, the phase is to winning
Obtain optimal treatment window phase.It clinically there is no quick detection human urine KIM-1 related reagent product both at home and abroad at present.
NGAL (neutrophil leucocyte gelatinase correlation apolipoprotein), be one by neutrophil leucocyte and part epithelial cell such as
Trace of albumin expressed by renal tubule.When ischemic or renal toxicity injury of kidney, NGAL is released to urine by kidney great expression
Liquid and blood plasma.NGAL contents are raised after damage occurs in 2h, and half-life period is 6h, can be fast in 1-2d after infection obtains controlling
Speed declines, and makes the injury of kidney mark of early stage and sensitivity.
TIMP-2 (tissue inhibitor of metalloproteinase -2), it is main to suppress after MMOL/LP-2 activity, Ischemic kieney injury,
TIMP-2 is induced high expression protection renal tubular epithelial and promotes injury repair.As new AKI marks, 2-3 phases AKI can be predicted
Occur.
IGFBP-7 (insulin-like growth factor binding protein-7), is a kind of secreted protein rich in cysteine,
IGF-1 receptor antagonists.IGFBP-7 rises change renal blood flow dynamics, aggravate kidney injury.IGFBP-7 contents can in urine
Predict that 2-3 phases AKI occur.TIMP-2 and IGBP-7 combinations can more Accurate Diagnosis AKI.
IL-18 (IL-18) is a kind of proinflammatory factor produced by mononuclear macrophage, is structurally and functionally all being belonged to
In IL-1 families.Research finds that IL-18 plays the part of important role in many kidney troubles, including ischemia-reperfusion, of the same race different
Body graft rejection, autoimmune state and malignant tumour.The inflammatory mediator discharged during as acute injury of kidney, as new
Injury of kidney mark is widely studied, and shows great diagnostic value.
PCT (Procalcitonin) is main by parafollicular cells of thyroid gland synthesis secretion.In bacterium infection, the macrophage of liver and
The lymphocyte and endocrine cell of monocyte, lung and intestinal tissue, endotoxin, tumor necrosis factor-alpha (TNF-α) and
The lower substantial amounts of PCT of synthesis secretion of the effect such as IL-6, causes blood-serum P CT levels significantly to raise.PCT is in whole body caused by bacterium infection
Property inflammatory reaction early stage (2~3h) can raise, 12~24h peaks after infection, and PCT concentration is with the infection order of severity in just
Correlation, infection recovers normal after disappearing, therefore early diagnosis to severe bacterial infections, judges coincident with severity degree of condition, prognosis, comments
The anti-infective curative effect of valency, instruct that all there is in terms of Antibiotics usage higher clinical value.
CTnI (cardiac muscle troponin I), in myocardial cell injury early stage, is free on intracytoplasmic cTnI quick releases and goes out
Come, level is raised in 4~6h in serum/plasma.As muscle fibril is constantly disintegrated destruction, the cTnI existed with fixed form is not
CTnI levels 8~14h after AMI generations reaches peak in disconnected release, serum/plasma, is down to after 1~2 week normal.Due to cTnI tools
With Cardiac-specific, the patient after 4h occurs for pectoralgia, directly can be detected using cTnI, and level rise has in its serum/plasma
The specificity of diagnosis, AMI early diagnosis can win the quality time for the treatment of patient.
Since occurring from label immunoassay technology, it has been widely used in medical science and biological study.Its principle
Exactly the specificity and sensitiveness of antigen-antibody reaction are combined with the accuracy of micro- spike, with colour developing or fluorescent material mark
Remember antibody, detection and localization is carried out to tissue or intracellular antigenic substance.In immunoassays, marked with enzyme or fluorescent material anti-
Body/antigen is required.Mechanical centrifugal method in general conventional method or other patents, enters to the fluorescent particle in labeling process
Row purifying, the labeling effciency of this method fluorescent particle is low, and the life activity of immunofluorescent particles is not also high.
And above-mentioned specific biomarker content in blood of human body or urine is relatively low, generally all in pik to receiving
Grams per milliliter, the immunofluorescence detection agent that is prepared using conventional method be extremely difficult to needed for high sensitive and high accurancy and precision
It is required that.And from the point of view of the clinical diagnostic applications scene of these marks, it typically belongs to clinical acute disease, it is badly in need of when very short
The completion high accurancy and precision of interior rapid sensitive is determined.
The content of the invention
There is shortcoming and defect in the present invention, the invention provides the specific high-affinity weight of one kind mark for current techniques
Kidney is damaged in fluorescent microsphere of group antibody and preparation method and application, particularly a kind of energy Quantitative detection people urine sample or blood sample
Hinder the related apolipoprotein (NGAL) of molecule -1 (KIM-1), neutrophil leucocyte gelatinase, insulin-like growth factor binding protein-7
(IGFBP-7), tissue inhibitor of metalloproteinase -2 (TIMP-2), interleukin 18 (IL-18), Procalcitonin (PCT), myocardium myo
The i.e. slotting fluoroscopic examination card of calcium protein I (cTnI).
Fluorescent microsphere of the specific high-affinity recombinant antibodies of a kind of mark that the present invention is provided and preparation method thereof is with answering
With being realized with following technical scheme：
A kind of preparation method of the fluorescent microsphere of the specific high-affinity recombinant antibodies of mark, comprises the following steps：
S1 is molten with the ethyl carbodiimide solution (EDC) of 1- (3- dimethylaminopropyls) -3 and n-hydroxysuccinimide
Liquid (NHS) is activated to fluorescent microsphere liquid respectively, obtains fluorescent microsphere activator mixture；Fluorescence in described fluorescent microsphere liquid
The particle diameter of microballoon is 250-350nm, solid content 1%；- 3 ethyl carbodiimide solution of the 1- (3- dimethylaminopropyls) are dense
Spend for 1-100mg/ml；The n-hydroxysuccinimide mixed solution concentration is 1-100mg/ml；The fluorescent microsphere liquid, 1-
(3- dimethylaminopropyls) -3 ethyl carbodiimide solution, the volume ratio of n-hydroxysuccinimide mixed solution are 100：1-
2：1-2.
The fluorescent microsphere activator mixture obtained in step S1 is put into bag filter by S2, in phosphate buffer PBS
Unnecessary 1- (3- dimethylaminopropyls) -3 ethyl carbodiimides of dialysis removing and n-hydroxysuccinimide, are obtained after dialysis
Fluorescent microsphere liquid；It is preferred that, the phosphoric acid buffer that heretofore described buffer solution is 10-100mmol/L PH 7.2-7.6
Liquid.
S3 will add amino-polyethyleneglycols carboxyl (NH in the fluorescent microsphere liquid after being dialysed in step S22- PEG-COOH),
After 37 DEG C of stirring reaction 1-3h, second of dialysis is carried out in faintly acid N- morpholino b acids (MES) cushioning liquid, it is unnecessary to remove
Amino-polyethyleneglycols carboxyl, obtain the fluorescent microsphere liquid after secondary dialysis；The molecular weight of the amino-polyethyleneglycols carboxyl is
1000~5000Da；It is preferred that, the amino-polyethyleneglycols carboxyl final concentration 10-100mmol/L of addition.It is preferred that, wherein buffering
Solution is 0.1mol/L pH4.5-6.1 N- morpholino b acid cushioning liquid.
Fluorescent microsphere liquid after the secondary dialysis that S4 will be obtained in step S3 is activated again, the fluorescence after being dialysed into S3
Liquid is added after 1- (3- dimethylaminopropyls) -3 ethyl carbodiimides (EDC) activation, adds specific high-affinity restructuring
Antibody adds bovine serum albumin(BSA) closing in reacting 8-12h at 2-8 DEG C, centrifugation, then by precipitation phosphate buffer
PBS is resuspended and (effect that can reach needs once or twice is resuspended), and centrifugation is redissolved liquid with fluorescent microsphere and is resuspended, that is, makes
The fluorescent microsphere of specific high-affinity recombinant antibodies must be marked.It is bright containing 0.1-1.5wt% that the fluorescent microsphere, which redissolves liquid,
Glue or BSA, 0.01-1wt% triton x-100,0.05-0.1wt% Sodium azides, 0.01-0.2mol/L pH 6.5-8.5 phosphoric acid
Salt buffer.Heretofore described phosphate buffer is 10-100mmol/L pH 7.2-7.6 phosphate buffer.It is preferred that
, when adding the bovine serum albumin(BSA) closing, make the final concentration of 0.5-1.5wt% of bovine serum albumin(BSA).
The whole gentle dialysis method using under specific system of the invention, rather than in general conventional method or other patents
Mechanical centrifugal method, the fluorescent particle in labeling process is purified, the benefit so done is to substantially increase fluorescent particle
Labeling effciency, and at utmost remain immunofluorescent particles life activity.
Wherein, the antibody in the step S4 is the anti-human KIM-1 monoclonal antibodies of mouse and goat anti-rabbit igg polyclonal antibody.
Wherein, the antibody in the step S4 is the anti-human NGAL of mouse and goat anti-rabbit igg polyclonal antibody.
Wherein, the antibody in the step S4 is the anti-human IGFBP-7 monoclonal antibodies of mouse and goat anti-rabbit igg Anti-TNF-α
Body.
Wherein, the antibody in the step S4 is mouse-anti-human T IMP-2 monoclonal antibodies and goat anti-rabbit igg polyclonal antibody.
Wherein, the antibody in the step S4 is the anti-human IL-18 of mouse and goat anti-rabbit igg.
Wherein, the antibody in the step S4 is the anti-human PCT of mouse and goat anti-rabbit igg.
Wherein, the antibody in the step S4 is the anti-human cTnI of mouse and goat anti-rabbit igg.
The fluorescent microsphere of the specific high-affinity recombinant antibodies of the obtained mark of the above method.
Fluorescence pad made from the fluorescent microsphere of the specific high-affinity recombinant antibodies of above-mentioned mark.
A kind of Quantitative detection KIM-1 detection card, including bottom plate and bottom plate are provided with sample pad, fluorescence pad, reaction
Pad, blotting paper；
Wherein, the fluorescence pad is made from the above method specificity anti-human KIM-1 of high-affinity recombinant murine will to be marked to resist
The fluorescent microsphere of body and the fluorescent microsphere of goat anti-rabbit igg antibody are by the fluorescent microsphere 1 in two kinds of fluorescent microsphere solution:1 equivalent is mixed
Close, be then added on polyester film pad, what freeze-drying was made；
Wherein, the reacting pad includes reacting pad body and which is provided with the anti-human KIM-1 monoclonals of another plant of mouse of coating resisting
Physical examination survey line and the nature controlling line for being coated with rabbit igg.
A kind of Quantitative detection NGAL detection card, including bottom plate and bottom plate are provided with sample pad, fluorescence pad, reaction
Pad, blotting paper；
Wherein, the fluorescence pad is the anti-human NGAL antibody of specific high-affinity recombinant murine will to be marked made from the above method
Fluorescent microsphere and goat anti-rabbit igg antibody fluorescent microsphere by the fluorescent microsphere 1 in two kinds of fluorescent microsphere solution:1 mixed in equal amounts,
Then it is added on polyester film pad, what freeze-drying was made；
Wherein, the reacting pad includes reaction pad body and which is provided with the coating anti-human NGAL recombinant monoclonals of another plant of mouse
Antibody detection line and the nature controlling line for being coated with rabbit igg.
A kind of Quantitative detection IGFBP-7 detection card, including bottom plate and bottom plate are provided with sample pad, fluorescence pad, anti-
Should pad, blotting paper；
Wherein, the fluorescence pad is the anti-human IGFBP-7 of specific high-affinity recombinant murine will to be marked made from the above method
The fluorescent microsphere of antibody and goat anti-rabbit igg antibody is by the fluorescent microsphere 1 in two kinds of fluorescent microsphere solution:1 mixed in equal amounts, Ran Houjia
On polyester film pad, what freeze-drying was made；
Wherein, the reacting pad includes reaction pad body and which is provided with another plant of anti-human IGFBP-7 of mouse of coating restructuring
Monoclonal antibody detection line and the nature controlling line for being coated with rabbit igg.
A kind of Quantitative detection TIMP-2 detection card, including bottom plate and bottom plate are provided with sample pad, fluorescence pad, reaction
Pad, blotting paper；
Wherein, the fluorescence pad is made from the above method specificity anti-human TIMP-2 of high-affinity recombinant murine will to be marked to resist
The fluorescent microsphere of body and goat anti-rabbit igg antibody is by the fluorescent microsphere 1 in two kinds of fluorescent microsphere solution:1 mixed in equal amounts, is then added in
On polyester film pad, what freeze-drying was made；
Wherein, the reacting pad includes reaction pad body and which is provided with another plant of mouse-anti-human T IMP-2 restructuring Dan Ke of coating
Grand antibody detection line and the nature controlling line for being coated with rabbit igg.
A kind of Quantitative detection IL-18 detection card, including bottom plate and bottom plate are provided with sample pad, fluorescence pad, reaction
Pad, blotting paper；
Wherein, the fluorescence pad is made from the above method specificity anti-human IL-18 of high-affinity recombinant murine will to be marked to resist
The fluorescent microsphere of body and goat anti-rabbit igg antibody is by the fluorescent microsphere 1 in two kinds of fluorescent microsphere solution:1 mixed in equal amounts, is then added in
On polyester film pad, what freeze-drying was made；
Wherein, the reacting pad includes reaction pad body and which is provided with the anti-human IL-18 restructuring Dan Ke of another plant of mouse of coating
Grand antibody detection line and the nature controlling line for being coated with rabbit igg.
A kind of Quantitative detection PCT detection card, including bottom plate and bottom plate provided with sample pad, fluorescence pad, reacting pad,
Blotting paper；
Wherein, the fluorescence pad is the anti-human PCT antibody of specific high-affinity recombinant murine will to be marked made from the above method
Fluorescent microsphere with goat anti-rabbit igg antibody is by the fluorescent microsphere 1 in two kinds of fluorescent microsphere solution:1 mixed in equal amounts, is then added in poly-
On ester film combination pad, what freeze-drying was made；
Wherein, the reacting pad includes reaction pad body and which is provided with the coating anti-human PCT recombinant monoclonals of another plant of mouse
Antibody detection line and the nature controlling line for being coated with rabbit igg.
A kind of Quantitative detection cTnI detection card, including bottom plate and bottom plate are provided with sample pad, fluorescence pad, reaction
Pad, blotting paper；
Wherein, the fluorescence pad is the anti-human cTnI antibody of specific high-affinity recombinant murine will to be marked made from the above method
Fluorescent microsphere with goat anti-rabbit igg antibody is by the fluorescent microsphere 1 in two kinds of fluorescent microsphere solution:1 mixed in equal amounts, is then added in poly-
On ester film combination pad, what freeze-drying was made；
Wherein, the reacting pad includes reaction pad body and which is provided with the coating anti-human cTnI recombinant monoclonals of another plant of mouse
Antibody detection line and the nature controlling line for being coated with rabbit igg.
Beneficial effects of the present invention：
1) fluorescent microsphere that the present invention is provided, according to conventional method, under the reaction that immunofluorescence microballoon can be significantly improved
Line so that reagent sensitivity can improve 10-100 times.Therefore mark content is common glimmering in the case of denier in sample to be tested
Light microballoon can not be detected accurately, and fluorescent microsphere of the present invention remains to obtain considerable fluorescence signal intensity so that immunofluorescence
The reactivity of particulate is greatly improved, so as to greatly improve reagent sensitivity and range of linearity isoreactivity energy.
2) what the present invention was provided, which inserts fluoroscopic examination card, can quantify detection KIM-1, NGAL, IGFBP-7, TIMP-2, IL-
18th, PCT, cTnI, it is easy to operate quick, quantitative result accurately and reliably, solve there is no at present Quantitative detection KIM-1,
The problem of NGAL, IGFBP-7, TIMP-2, IL-18, PCT, cTnI.It is suitable for hygiene medical treatment mechanisms at different levels, including township hospital
Hygienic clinic etc., with important social effect.
Brief description of the drawings
Fig. 1 is detection card structure schematic diagram：1 sample pad, 2 fluorescence pads, 3 reacting pads, 4 detection lines, 5 nature controlling lines, 6 water suctions
Paper, 7 bottom plates.
Fig. 2 contrasts for the KIM-1 calibration curves of embodiment 1, comparative example 1 and comparative example 2.
Fig. 3 is the KIM-1 measured values of embodiment 1, comparative example 1 and comparative example 2 and the regression curve of theoretical value.
Fig. 4 contrasts for the NGAL calibration curves of embodiment 2, comparative example 3 and comparative example 4
Fig. 5 is the NGAL measured values of embodiment 2, comparative example 3 and comparative example 4 and the regression curve of theoretical value.
Fig. 6 contrasts for the IGFBP-7 calibration curves of embodiment 3, comparative example 5 and comparative example 6
Fig. 7 is the IGFBP-7 measured values of embodiment 3, comparative example 5 and comparative example 6 and the regression curve of theoretical value.
Fig. 8 is the TIMP-2 calibration curves of embodiment 4, comparative example 7 and comparative example 8
Fig. 9 is the TIMP-2 measured values of embodiment 4, comparative example 7 and comparative example 8 and the regression curve of theoretical value.
Figure 10 is the IL-18 calibration curves of embodiment 5, comparative example 9 and comparative example 10
Figure 11 is the IL-18 measured values of embodiment 5, comparative example 9 and comparative example 10 and the regression curve of theoretical value.
Figure 12 is the PCT calibration curves of embodiment 6, comparative example 11 and comparative example 12
Figure 13 is the PCT measured values of embodiment 6, comparative example 11 and comparative example 12 and the regression curve of theoretical value.
Figure 14 is the cTnI calibration curves of embodiment 7, comparative example 13 and comparative example 14
Figure 15 is the cTnI measured values of embodiment 7, comparative example 13 and comparative example 14 and the regression curve of theoretical value.
Embodiment
With reference to embodiment, the invention will be further described：
Embodiment 1
In embodiment 1 and comparative example 1,2：
Fluorescent microsphere liquid：Particle diameter 300nm, solid content 1%；
EDC solution：50mg/ml 1- (3- dimethylaminopropyls) -3 ethyl carbodiimides (EDC),；
NHS solution：50mg/ml n-hydroxysuccinimides (NHS)；
MES buffer solutions：0.1M MES PH 5N- morpholino b acids (MES)；
PBS：The phosphate (PBS) of 0.05M PBS PH 7.4；
Amino-polyethyleneglycols carboxyl (NH2- PEG-COOH), molecular weight is 2000Dalton.
Fluorescent microsphere redissolves liquid：Containing 0.2wt%BSA, 0.1wt% Qula X-100,0.08wt% Sodium azide, 0.1M, PH
8.0 phosphate buffer.
Prepare the fluoroscopic examination card of KIM-1 in Quantitative detection human urine
1st, the preparation of KIM-1 antibody labelings fluorescent microsphere：
1.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
1.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
Fluorescent microsphere solution after above-mentioned activation is loaded bag filter (molecular cut off 30KD) by 1.3, in 200ml PBS
In 2~8 DEG C of dialysis in buffer solution, unnecessary reaction residue is removed；
1.4 add amino-polyethyleneglycols carboxyl, amino-polyethyleneglycols carboxyl into the fluorescent microsphere solution after above-mentioned dialysis
Final concentration 50mM, 37 DEG C are reacted 1~3 hour；
Above-mentioned reacted microspheres solution is loaded bag filter by 1.5, is dialysed in 200ml MES buffer solutions, it is unnecessary to remove
Reaction residue；
1.6 add EDC solution into the fluorescent microsphere solution after above-mentioned dialysis (adds 0.1ml per 1ml fluorescent microsphere liquid
EDC solution), room temperature priming reaction 15 minutes；
1.7 fluorescence for taking the anti-human KIM-1 monoclonal antibodies of 1mg mouse (8.0mg/ml takes 125 μ l) to add after above-mentioned activation are micro-
In ball solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
1.8 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
1.9 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.10 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
1.11 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.12 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
2nd, the preparation of goat anti-rabbit antibody mark fluorescent microballoon：
2.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
2.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
Fluorescent microsphere solution after above-mentioned activation is loaded bag filter (molecular cut off 30KD) by 2.3, in 200ml PBS
In 2~8 DEG C of dialysis in buffer solution, unnecessary reaction residue is removed；
2.4 add amino-polyethyleneglycols carboxyl, amino-polyethyleneglycols carboxyl into the fluorescent microsphere solution after above-mentioned dialysis
Final concentration 50mM, 37 DEG C are reacted 1~3 hour；
Above-mentioned reacted microspheres solution is loaded bag filter by 2.5, is dialysed in 200ml MES buffer solutions, it is unnecessary to remove
Reaction residue；
2.6 add EDC solution into the fluorescent microsphere solution after above-mentioned dialysis (adds 0.1ml per 1ml fluorescent microsphere liquid
EDC solution), room temperature priming reaction 15 minutes；
2.7 take the fluorescent microsphere that 0.5mg goat-antis rabbit polyclonal antibody (10.0mg/ml takes 50 μ l) is added after above-mentioned activation
In solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
2.8 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
2.9 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.10 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
2.11 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.12 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
3 prepare the fluoroscopic examination card of KIM-1 in Quantitative detection human urine
3.1 prepare sample pad：Using glass fibre as urine specimen pad, immersion containing 1% skimmed milk power or BSA,
0.1% Tween-20, the phosphate buffers of 0.1M PH 7.40, drying at room temperature, hermetic bag hermetically drying are preserved.
3.2 prepare fluorescence pad：It is anti-human that the specific high-affinity recombinant murine of mark is diluted respectively with fluorescent microsphere redissolution liquid
The fluorescent microsphere of KIM-1 monoclonal antibodies and the fluorescent microsphere of goat anti-rabbit igg polyclonal antibody, 1 is pressed by the microballoon after mark:1
Volume ratio is mixed, and is then added on polyester film pad, is freeze-dried, and hermetic bag hermetically drying is preserved.
3.3 prepare reacting pad：
It is coated with buffer solution：0.1M PH 8 phosphate, 5wt% sucrose, 0.05wt% Tween-20.
Nitrocellulose filter：Outsourcing
Prepare reacting pad:It is 1.0mg/ that the anti-human KIM-1 monoclonal antibodies of another plant of mouse to concentration is diluted with coating buffer solution
Ml, with coating buffer solution dilution rabbit igg, the rabbit igg concentration of dilution is 1.0mg/ml.Then existed using two kinds of antibody after dilution
Line parallel successively forms detection line, nature controlling line on nitrocellulose filter.
3.4 blotting papers, bottom plate outsourcing.
3.5 prepare the fluoroscopic examination card of KIM-1 in Quantitative detection human urine：By sample pad, fluorescence pad, reacting pad,
Blotting paper and bottom plate are combined into as shown in Figure 1.
Comparative example 1
1st, the preparation of KIM-1 antibody labelings fluorescent microsphere：
1.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
1.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
1.3 20000 revs/min of centrifugations are centrifuged 30 minutes；
1.4 remove supernatant, and precipitation is resuspended with 1ml PBSs, and makes microballoon within ultrasonically treated 10 minutes with Ultrasound Instrument
Solution is uniformly dispersed；
1.5 add amino-polyethyleneglycols carboxyl, amino-polyethyleneglycols into the fluorescent microsphere solution after above-mentioned centrifugal treating
Carboxyl final concentration 50mM, 37 DEG C are reacted 1~3 hour；
Above-mentioned reacted microspheres solution is loaded bag filter by 1.6, is dialysed in 200ml MES buffer solutions, it is unnecessary to remove
Reaction residue；
1.7 add EDC solution into the fluorescent microsphere solution after above-mentioned dialysis (adds 0.1ml per 1ml fluorescent microsphere liquid
EDC solution), room temperature priming reaction 15 minutes；
1.8 fluorescence for taking the anti-human KIM-1 monoclonal antibodies of 1mg mouse (8.0mg/ml takes 125 μ l) to add after above-mentioned activation are micro-
In ball solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
1.9 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
1.10 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.11 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
1.12 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.13 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
2nd, the preparation of goat anti-rabbit antibody mark fluorescent microballoon：
2.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
2.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
2.3 20000 revs/min of centrifugations are centrifuged 30 minutes；
2.4 remove supernatant, and precipitation is resuspended with 1ml PBSs, and makes microballoon within ultrasonically treated 10 minutes with Ultrasound Instrument
Solution is uniformly dispersed；
2.5 add amino-polyethyleneglycols carboxyl, amino-polyethyleneglycols into the fluorescent microsphere solution after above-mentioned centrifugal treating
Carboxyl final concentration 50mM, 37 DEG C are reacted 1~3 hour；
Above-mentioned reacted microspheres solution is loaded bag filter by 2.6, is dialysed in 200ml MES buffer solutions, it is unnecessary to remove
Reaction residue；
2.7 add EDC solution into the fluorescent microsphere solution after above-mentioned dialysis (adds 0.1ml per 1ml fluorescent microsphere liquid
EDC solution), room temperature priming reaction 15 minutes；
2.8 take the fluorescent microsphere that 0.5mg goat-antis rabbit polyclonal antibody (10.0mg/ml takes 50 μ l) is added after above-mentioned activation
In solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
2.9 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
2.10 by above-mentioned solution centrifugal, 2~8 DEG C 20000 leaves the heart 15 minutes；
2.11 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
2.12 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.13 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
3.1 prepare the fluoroscopic examination card of KIM-1 in detection human urine：Described in method be the same as Example 1.
Comparative example 2
1st, the preparation of KIM-1 antibody labelings fluorescent microsphere：
1.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
1.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
Fluorescent microsphere solution after above-mentioned activation is loaded bag filter (molecular cut off 30KD) by 1.3, in 200ml PBS
In 2~8 DEG C of dialysis in buffer solution, unnecessary reaction residue is removed；
1.4 fluorescence for taking the anti-human KIM-1 monoclonal antibodies of 1mg mouse (8.0mg/ml takes 125 μ l) to add after above-mentioned activation are micro-
In ball solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
1.5 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
1.6 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.7 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
1.8 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.9 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
2nd, the preparation of goat anti-rabbit antibody mark fluorescent microballoon：
2.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
2.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
Fluorescent microsphere solution after above-mentioned activation is loaded bag filter (molecular cut off 30KD) by 2.3, in 200ml PBS
In 2~8 DEG C of dialysis in buffer solution, unnecessary reaction residue is removed；
2.4 take the fluorescent microsphere that 0.5mg goat-antis rabbit polyclonal antibody (10.0mg/ml takes 50 μ l) is added after above-mentioned activation
In solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
2.5 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
2.6 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.7 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
2.8 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.9 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
3.1 prepare the fluoroscopic examination card of KIM-1 in detection human urine：Described in method be the same as Example 1.
Embodiment 1, comparative example 1, the test and comparison result of comparative example 2 are as follows：
KIM-1 immunofluorescence detection agent cards prepared by embodiment 1, comparative example 1, the method for comparative example 2 are respectively adopted, survey
Difference of the detection reagent on calibration work curve prepared by different process method is compared in examination, and in sensitivity for analysis, line
Difference in property scope main performance index.
A uses the calibration results and its working curve of gradient concentration KIM-1 calibration objects as shown in Figure 2：
From the calibration results, the calibration results signal value of embodiment 1 is apparently higher than comparing example 1 and comparative example 2；Embodiment
Each concentration point signaling zone is indexed apparently higher than comparative example 1 and comparative example 2. on 1 working curve
B sensitivity for analysis test results
Compare fluorescence signal between 0.5ng/ml samples and blank sample (0ng/ml) poor
Δ difference：The same KIM-1 low concentration samples (0.5ng/ml) of test, the signal sensitivity of embodiment 1 apparently higher than
Comparative example 1 and comparative example 2.
C range of linearity test results
The KIM-1 detection reagents card prepared respectively using three kinds of different process methods detects high, medium and low various concentrations scope
Concentration known sample, compare the recurrence difference of measured value and theoretical value.
The regression result can as shown in Figure 3, and the range of linearity performance of embodiment 1 is substantially better than comparative example 1 and comparative example 2.
Embodiment 2
In embodiment 2 and comparative example 3,4：
Fluorescent microsphere liquid：Particle diameter 300nm, solid content 1%；
EDC solution：50mg/ml 1- (3- dimethylaminopropyls) -3 ethyl carbodiimides (EDC),；
NHS solution：50mg/ml n-hydroxysuccinimides (NHS)；
MES buffer solutions：0.1M MES PH 5N- morpholino b acids (MES)；
PBS：The phosphate (PBS) of 0.05M PBS PH 7.4；
Amino-polyethyleneglycols carboxyl (NH2- PEG-COOH), molecular weight is 2000Dalton.
Fluorescent microsphere redissolves liquid：Containing 0.2wt%BSA, 0.1wt% Qula X-100,0.08wt% Sodium azide, 0.1M, PH
8.0 phosphate buffer.
Prepare the fluoroscopic examination card of NGAL in Quantitative detection blood
1st, the preparation of NGAL antibody labelings fluorescent microsphere：
1.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
1.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
Fluorescent microsphere solution after above-mentioned activation is loaded bag filter (molecular cut off 30KD) by 1.3, in 200ml PBS
In 2~8 DEG C of dialysis in buffer solution, unnecessary reaction residue is removed；
1.4 add amino-polyethyleneglycols carboxyl, amino-polyethyleneglycols carboxyl into the fluorescent microsphere solution after above-mentioned dialysis
Final concentration 50mM, 37 DEG C are reacted 1~3 hour；
Above-mentioned reacted microspheres solution is loaded bag filter by 1.5, is dialysed in 200ml buffer solutions, is removed unnecessary anti-
Answer residue；
1.6 add EDC solution into the fluorescent microsphere solution after above-mentioned dialysis (adds 0.1ml per 1ml fluorescent microsphere liquid
EDC solution), room temperature priming reaction 15 minutes；
1.7 fluorescent microspheres for taking 0.8mg NGAL monoclonal antibodies (5.8mg/ml takes 138 μ l) to add after above-mentioned activation are molten
In liquid, mix immediately, 2~8 DEG C are reacted 8~12 hours；
1.8 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
1.9 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.10 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
1.11 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.12 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
2nd, the preparation of goat anti-rabbit antibody mark fluorescent microballoon：
2.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
2.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
Fluorescent microsphere solution after above-mentioned activation is loaded bag filter (molecular cut off 30KD) by 2.3, in 200ml PBS
In 2~8 DEG C of dialysis in buffer solution, unnecessary reaction residue is removed；
2.4 add amino-polyethyleneglycols carboxyl, amino-polyethyleneglycols carboxyl into the fluorescent microsphere solution after above-mentioned dialysis
Final concentration 50mM, 37 DEG C are reacted 1~3 hour；
Above-mentioned reacted microspheres solution is loaded bag filter by 2.5, is dialysed in 200ml MES buffer solutions, it is unnecessary to remove
Reaction residue；
2.6 add EDC solution into the fluorescent microsphere solution after above-mentioned dialysis (adds 0.1ml per 1ml fluorescent microsphere liquid
EDC solution), room temperature priming reaction 15 minutes；
2.7 take the fluorescent microsphere that 0.5mg goat-antis rabbit polyclonal antibody (10.0mg/ml takes 50 μ l) is added after above-mentioned activation
In solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
2.8 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
2.9 by above-mentioned solution centrifugal, 2~8 DEG C 20000 leaves the heart 15 minutes；
2.10 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
2.11 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.12 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
3 prepare the fluoroscopic examination card of NGAL in Quantitative detection human blood
3.1 prepare sample pad：Using glass fibre as urine specimen pad, immersion containing 1% skimmed milk power or BSA,
0.1% Tween-20, the phosphate buffers of 0.1M 7.40, drying at room temperature, hermetic bag hermetically drying are preserved.
3.2 prepare fluorescence pad：Liquid, which is redissolved, with fluorescent microsphere dilutes the specific high-affinity restructuring NGAL Dan Ke of mark respectively
The fluorescent microsphere of grand antibody and the fluorescent microsphere of goat anti-rabbit igg polyclonal antibody, 1 is pressed by the microballoon after mark:1 volume ratio is mixed
Close, be then added on polyester film pad, be freeze-dried, hermetic bag hermetically drying is preserved.
3.3 prepare reacting pad：
It is coated with buffer solution：0.1M PH8 phosphate, 5wt% sucrose, 0.05wt% Tween-20.
Nitrocellulose filter：Outsourcing
Prepare reacting pad:It is 1.0mg/ml that another plant of NGAL monoclonal antibodies to concentration is diluted with coating buffer solution, with bag
Rabbit igg is diluted by buffer solution respectively, the IgG concentration of dilution is 1.0mg/ml.Then using two kinds of antibody after dilution in nitric acid
Line parallel successively forms detection line, nature controlling line on cellulose membrane.
3.4 blotting papers, bottom plate outsourcing.
3.5 prepare the fluoroscopic examination card of NGAL in Quantitative detection human blood：By sample pad, fluorescence pad, reacting pad,
Blotting paper and bottom plate are combined into as shown in Figure 1.
Comparative example 3
1st, the preparation of NGAL antibody labelings fluorescent microsphere：
1.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
1.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
1.3 20000 revs/min of centrifugations are centrifuged 30 minutes；
1.4 remove supernatant, and precipitation is resuspended with 1ml PBSs, and makes microballoon within ultrasonically treated 10 minutes with Ultrasound Instrument
Solution is uniformly dispersed；
1.5 add amino-polyethyleneglycols carboxyl, amino-polyethyleneglycols into the fluorescent microsphere solution after above-mentioned centrifugal treating
Carboxyl final concentration 50mM, 37 DEG C are reacted 1~3 hour；
Above-mentioned reacted microspheres solution is loaded bag filter by 1.6, is dialysed in 200ml MES buffer solutions, it is unnecessary to remove
Reaction residue；
1.7 add EDC solution into the fluorescent microsphere solution after above-mentioned dialysis (adds 0.1ml per 1ml fluorescent microsphere liquid
EDC solution), room temperature priming reaction 15 minutes；
1.8 fluorescent microspheres for taking 0.8mg NGAL monoclonal antibodies (5.8mg/ml takes 138 μ l) to add after above-mentioned activation are molten
In liquid, mix immediately, 2~8 DEG C are reacted 8~12 hours；
1.9 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
1.10 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.11 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
1.12 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.13 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
2nd, the preparation of goat anti-rabbit antibody mark fluorescent microballoon：
2.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
2.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
2.3 20000 revs/min of centrifugations are centrifuged 30 minutes；
2.4 remove supernatant, and precipitation is resuspended with 1ml PBSs, and makes microballoon within ultrasonically treated 10 minutes with Ultrasound Instrument
Solution is uniformly dispersed；
2.5 add amino-polyethyleneglycols carboxyl, amino-polyethyleneglycols into the fluorescent microsphere solution after above-mentioned centrifugal treating
Carboxyl final concentration 50mM, 37 DEG C are reacted 1~3 hour；
Above-mentioned reacted microspheres solution is loaded bag filter by 2.6, is dialysed in 200ml MES buffer solutions, it is unnecessary to remove
Reaction residue；
2.7 add EDC solution into the fluorescent microsphere solution after above-mentioned dialysis (adds 0.1ml per 1ml fluorescent microsphere liquid
EDC solution), room temperature priming reaction 15 minutes；
2.8 take the fluorescent microsphere that 0.5mg goat-antis rabbit polyclonal antibody (10.0mg/ml takes 50 μ l) is added after above-mentioned activation
In solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
2.9 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
2.10 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.11 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
2.12 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.13 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
3.1 prepare the fluoroscopic examination card of NGAL in detection blood：Described in method be the same as Example 2.
Comparative example 4
1st, the preparation of NGAL antibody labelings fluorescent microsphere：
1.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
1.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
Fluorescent microsphere solution after above-mentioned activation is loaded bag filter (molecular cut off 30KD) by 1.3, in 200ml PBS
In 2~8 DEG C of dialysis in buffer solution, unnecessary reaction residue is removed；
1.5 take the fluorescence that the anti-human NGAL monoclonal antibodies of 0.8mg mouse (5.8mg/ml takes 138 μ l) are added after above-mentioned activation
In microspheres solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
1.6 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
1.7 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.8 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
1.9 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.10 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
2nd, the preparation of goat anti-rabbit antibody mark fluorescent microballoon：
2.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
2.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
Fluorescent microsphere solution after above-mentioned activation is loaded bag filter (molecular cut off 30KD) by 2.3, in 200ml PBS
In 2~8 DEG C of dialysis in buffer solution, unnecessary reaction residue is removed；
2.4 take the fluorescent microsphere that 0.5mg goat-antis rabbit polyclonal antibody (10.0mg/ml takes 50 μ l) is added after above-mentioned activation
In solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
2.5 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
2.6 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.7 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
2.8 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.9 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
3.1 prepare the fluoroscopic examination card of NGAL in detection human urine：Described in method be the same as Example 2.
Test data：
Embodiment 2, comparative example 3, the test and comparison result of comparative example 4 are as follows：
NGAL immunofluorescence detection agent cards prepared by embodiment 2, comparative example 3, the method for comparative example 4 are respectively adopted, survey
Difference of the detection reagent on calibration work curve prepared by different process method is compared in examination, and in sensitivity for analysis, line
Difference in property scope main performance index：
A uses gradient concentration NGAL calibration objects (1:100 dilution) the calibration results and its working curve as shown in Figure 4
From the calibration results, the calibration results signal value of embodiment 2 is apparently higher than comparing example 3 and comparative example 4；Embodiment
Each concentration point signaling zone is indexed apparently higher than comparative example 3 and comparative example 4 on 2 working curves
B. sensitivity for analysis
Compare fluorescence signal between 50ng/ml samples and blank sample (0ng/ml) poor
Δ difference：The same NGAL low concentration samples (0.5ng/ml) of test, the signal sensitivity of embodiment 2 is apparently higher than right
Ratio 3 and comparative example 4.
C. the range of linearity
The NGAL detection reagents card prepared respectively using three kinds of different process methods detects high, medium and low various concentrations scope
Concentration known sample, compare the recurrence difference of measured value and theoretical value.
The regression result can as shown in Figure 5, and the range of linearity performance of embodiment 2 is substantially better than comparative example 3 and comparative example 4.
Embodiment 3
In embodiment 3 and comparative example 5,6：
Fluorescent microsphere liquid：Particle diameter 250nm, solid content 1%；
EDC solution：50mg/ml 1- (3- dimethylaminopropyls) -3 ethyl carbodiimides (EDC),；
NHS solution：50mg/ml n-hydroxysuccinimides (NHS)；
MES buffer solutions：0.1M MES PH 5N- morpholino b acids (MES)；
PBS：The phosphate (PBS) of 0.05M PBS PH 7.4；
Amino-polyethyleneglycols carboxyl (NH2- PEG-COOH), molecular weight is 1000Dalton.
Fluorescent microsphere redissolves liquid：Containing 0.2wt%BSA, 0.1wt% Qula X-100,0.08wt% Sodium azide, 0.1M, PH
8.0 phosphate buffer.
Prepare the fluoroscopic examination card of IGFBP-7 in Quantitative detection urine
1st, the preparation of IGFBP-7 antibody labelings fluorescent microsphere：
1.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
1.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
Fluorescent microsphere solution after above-mentioned activation is loaded bag filter (molecular cut off 30KD) by 1.3, in 200ml PBS
In 2~8 DEG C of dialysis in buffer solution, unnecessary reaction residue is removed；
1.4 add amino-polyethyleneglycols carboxyl, amino-polyethyleneglycols carboxyl into the fluorescent microsphere solution after above-mentioned dialysis
Final concentration 50mM, 37 DEG C are reacted 1~3 hour；
Above-mentioned reacted microspheres solution is loaded bag filter by 1.5, is dialysed in 200ml MES buffer solutions, it is unnecessary to remove
Reaction residue；
1.6 add EDC solution into the fluorescent microsphere solution after above-mentioned dialysis (adds 0.1ml per 1ml fluorescent microsphere liquid
EDC solution), room temperature priming reaction 15 minutes；
1.7 to take the anti-human IGFBP-7 monoclonal antibodies of 0.6mg mouse (5.3mg/ml takes 113 μ l) to add glimmering after above-mentioned activation
In light microspheres solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
1.8 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
1.9 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.10 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
1.11 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.12 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
2nd, the preparation of goat anti-rabbit antibody mark fluorescent microballoon：
2.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
2.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
Fluorescent microsphere solution after above-mentioned activation is loaded bag filter (molecular cut off 30KD) by 2.3, in 200ml PBS
In 2~8 DEG C of dialysis in buffer solution, unnecessary reaction residue is removed；
2.4 add amino-polyethyleneglycols carboxyl, amino-polyethyleneglycols carboxyl into the fluorescent microsphere solution after above-mentioned dialysis
Final concentration 10mM, 37 DEG C are reacted 1~3 hour；
Above-mentioned reacted microspheres solution is loaded bag filter by 2.5, is dialysed in 200ml MES buffer solutions, it is unnecessary to remove
Reaction residue；
2.6 add EDC solution into the fluorescent microsphere solution after above-mentioned dialysis (adds 0.1ml per 1ml fluorescent microsphere liquid
EDC solution), room temperature priming reaction 15 minutes；
2.7 take the fluorescent microsphere that 0.5mg goat-antis rabbit polyclonal antibody (10.0mg/ml takes 50 μ l) is added after above-mentioned activation
In solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
2.8 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
2.9 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.10 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
2.11 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.12 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
3 prepare the fluoroscopic examination card of IGFBP-7 in Quantitative detection human blood
3.1 prepare sample pad：Using glass fibre as urine specimen pad, immersion containing 1% skimmed milk power or BSA,
0.1% Tween-20, the phosphate buffers of 0.1MOL/L PH 7.40, drying at room temperature, hermetic bag hermetically drying are preserved.
3.2 prepare fluorescence pad：Liquid, which is redissolved, with fluorescent microsphere dilutes the specific high-affinity restructuring IGFBP-7 of mark respectively
The fluorescent microsphere of monoclonal antibody and the fluorescent microsphere of goat anti-rabbit igg polyclonal antibody, 1 is pressed by the microballoon after mark:1 volume ratio
Mixing, is then added on polyester film pad, is freeze-dried, and hermetic bag hermetically drying is preserved.
3.3 prepare reacting pad：
It is coated with buffer solution：0.1MOL/L PH 8 phosphate, 5wt% sucrose, 0.05wt% Tween-20.Dilution
IGFBP-7 antibody concentrations are 1.5mg/ml, and the IgG concentration of dilution is 1.0mg/ml.
Nitrocellulose filter：Outsourcing
Prepare reacting pad:Diluting the anti-human IGFBP-7 monoclonal antibodies of another plant of mouse to concentration respectively with coating buffer solution is
1.5mg/ml, with coating buffer solution dilution rabbit igg, the IgG concentration of dilution is 1.0mg/ml.Then resisted using two kinds after dilution
Body line parallel successively on nitrocellulose filter forms detection line, nature controlling line.
3.4 blotting papers, bottom plate outsourcing.
3.5 prepare the fluoroscopic examination card of IGFBP-7 in Quantitative detection human urine：Will be by sample pad, fluorescence pad, anti-
Should pad, blotting paper be combined into bottom plate it is as shown in Figure 1.
Comparative example 5
1st, the preparation of IGFBP-7 antibody labelings fluorescent microsphere：
1.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
1.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
1.3 20000 revs/min of centrifugations are centrifuged 30 minutes；
1.4 remove supernatant, and precipitation is resuspended with 1ml PBSs, and makes microballoon within ultrasonically treated 10 minutes with Ultrasound Instrument
Solution is uniformly dispersed；
1.5 add amino-polyethyleneglycols carboxyl, amino-polyethyleneglycols into the fluorescent microsphere solution after above-mentioned centrifugal treating
Carboxyl final concentration 10mM, 37 DEG C are reacted 1~3 hour；
Above-mentioned reacted microspheres solution is loaded bag filter by 1.6, is dialysed in 200ml MES buffer solutions, it is unnecessary to remove
Reaction residue；
1.7 add EDC solution into the fluorescent microsphere solution after above-mentioned dialysis (adds 0.1ml per 1ml fluorescent microsphere liquid
EDC solution), room temperature priming reaction 15 minutes；
1.8 fluorescence for taking 0.6mg IGFBP-7 monoclonal antibodies (5.3mg/ml takes 113 μ l) to add after above-mentioned activation are micro-
In ball solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
1.9 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
1.10 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.11 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
1.12 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.13 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
2nd, the preparation of goat anti-rabbit antibody mark fluorescent microballoon：
2.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
2.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
2.3 20000 revs/min of centrifugations are centrifuged 30 minutes；
2.4 remove supernatant, and precipitation is resuspended with 1ml PBSs, and makes microballoon within ultrasonically treated 10 minutes with Ultrasound Instrument
Solution is uniformly dispersed；
2.5 add amino-polyethyleneglycols carboxyl, amino-polyethyleneglycols into the fluorescent microsphere solution after above-mentioned centrifugal treating
Carboxyl final concentration 10mM, 37 DEG C are reacted 1~3 hour；
Above-mentioned reacted microspheres solution is loaded bag filter by 2.6, is dialysed in 200ml MES buffer solutions, it is unnecessary to remove
Reaction residue；
2.7 add EDC solution into the fluorescent microsphere solution after above-mentioned dialysis (adds 0.1ml per 1ml fluorescent microsphere liquid
EDC solution), room temperature priming reaction 15 minutes；
2.8 take the fluorescent microsphere that 0.5mg goat-antis rabbit polyclonal antibody (10.0mg/ml takes 50 μ l) is added after above-mentioned activation
In solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
2.9 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
2.10 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.11 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
2.12 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.13 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
3.1 prepare the IGFBP-7 fluoroscopic examination cards in detection urine：Described in method be the same as Example 3.
Comparative example 6
1st, the preparation of IGFBP-7 antibody labelings fluorescent microsphere：
1.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
1.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
Fluorescent microsphere solution after above-mentioned activation is loaded bag filter (molecular cut off 30KD) by 1.3, in 200ml PBS
In 2~8 DEG C of dialysis in buffer solution, unnecessary reaction residue is removed；
1.4 to take the anti-human IGFBP-7 monoclonal antibodies of 0.6mg mouse (5.3mg/ml takes 113 μ l) to add glimmering after above-mentioned activation
In light microspheres solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
1.5 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
1.6 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.7 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
1.8 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.9 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
2nd, the preparation of goat anti-rabbit antibody mark fluorescent microballoon：
2.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
2.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
Fluorescent microsphere solution after above-mentioned activation is loaded bag filter (molecular cut off 30KD) by 2.3, in 200ml PBS
In 2~8 DEG C of dialysis in buffer solution, unnecessary reaction residue is removed；
2.4 take the fluorescent microsphere that 0.5mg goat-antis rabbit polyclonal antibody (10.0mg/ml takes 50 μ l) is added after above-mentioned activation
In solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
2.5 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
2.6 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.7 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
2.8 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.9 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
3.1 prepare the fluoroscopic examination card of IGFBP-7 in detection human urine：Described in method be the same as Example 3.
Test data：
Embodiment 3, comparative example 5, the test and comparison result of comparative example 6 are as follows：
IGFBP-7 immunofluorescence detection agent cards prepared by embodiment 3, comparative example 5, the method for comparative example 6 are respectively adopted,
Difference of the detection reagent on calibration work curve prepared by test and comparison different process method, and sensitivity for analysis,
Difference in range of linearity main performance index：
A uses gradient concentration IGFBP-7 calibration objects (1:100 dilution) the calibration results and its working curve as shown in Figure 6
From the calibration results, the calibration results signal value of embodiment 3 is apparently higher than comparing example 5 and comparative example 6；Embodiment
Each concentration point signaling zone is indexed apparently higher than comparative example 5 and comparative example 6 on 5 working curves.
B sensitivity for analysis
Compare fluorescence signal between 10ng/ml samples and blank sample (0ng/ml) poor
Δ difference：The same IGFBP-7 low concentration samples (10ng/ml) of test, the signal sensitivity of embodiment 3 apparently higher than
Comparative example 5 and comparative example 6.
The C ranges of linearity
The IGFBP-7 detection reagents card prepared respectively using three kinds of different process methods detects high, medium and low various concentrations model
The concentration known sample enclosed, compares the recurrence difference of measured value and theoretical value, as shown in Figure 7.
The regression result is visible, and the range of linearity performance of embodiment 3 is with being substantially better than comparative example 5 and comparative example 6.
Embodiment 4
In embodiment 4 and comparative example 7,8：
Fluorescent microsphere liquid：Particle diameter 350nm, solid content 1%；
EDC solution：50mg/ml 1- (3- dimethylaminopropyls) -3 ethyl carbodiimides (EDC),；
NHS solution：50mg/ml n-hydroxysuccinimides (NHS)；
MES buffer solutions：0.1M MES PH 5N- morpholino b acids (MES)；
PBS：The phosphate (PBS) of 0.05M PBS PH 7.4；
Amino-polyethyleneglycols carboxyl (NH2- PEG-COOH), molecular weight is 5000Dalton.
Fluorescent microsphere redissolves liquid：Containing 0.2wt%BSA, 0.1wt% Qula X-100,0.08wt% Sodium azide, 0.1M, PH
8.0 phosphate buffer.
Prepare the fluoroscopic examination card of TIMP-2 in Quantitative detection urine
1st, the preparation of TIMP-2 antibody labelings fluorescent microsphere：
1.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
1.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
Fluorescent microsphere solution after above-mentioned activation is loaded bag filter (molecular cut off 30KD) by 1.3, in 200ml PBS
In 2~8 DEG C of dialysis in buffer solution, unnecessary reaction residue is removed；
1.4 add amino-polyethyleneglycols carboxyl, amino-polyethyleneglycols carboxyl into the fluorescent microsphere solution after above-mentioned dialysis
Final concentration 100mM, 37 DEG C are reacted 1~3 hour；
Above-mentioned reacted microspheres solution is loaded bag filter by 1.5, is dialysed in 200ml MES buffer solutions, it is unnecessary to remove
Reaction residue；
1.6 add EDC solution into the fluorescent microsphere solution after above-mentioned dialysis (adds 0.1ml per 1ml fluorescent microsphere liquid
EDC solution), room temperature priming reaction 15 minutes；
1.7 take the fluorescence that 0.6mg mouse-anti-human T IMP-2 monoclonal antibodies (6.9mg/ml takes 87 μ l) are added after above-mentioned activation
In microspheres solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
1.8 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
1.9 by above-mentioned solution centrifugal, 2~8 DEG C 20000 leaves the heart 15 minutes；
1.10 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
1.11 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.12 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
2nd, the preparation of goat anti-rabbit antibody mark fluorescent microballoon：
2.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
2.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
Fluorescent microsphere solution after above-mentioned activation is loaded bag filter (molecular cut off 30KD) by 2.3, in 200ml PBS
In 2~8 DEG C of dialysis in buffer solution, unnecessary reaction residue is removed；
2.4 add amino-polyethyleneglycols carboxyl, amino-polyethyleneglycols carboxyl into the fluorescent microsphere solution after above-mentioned dialysis
Final concentration 100mM, 37 DEG C are reacted 1~3 hour；
Above-mentioned reacted microspheres solution is loaded bag filter by 2.5, is dialysed in 200ml MES buffer solutions, it is unnecessary to remove
Reaction residue；
2.6 add EDC solution into the fluorescent microsphere solution after above-mentioned dialysis (adds 0.1ml per 1ml fluorescent microsphere liquid
EDC solution), room temperature priming reaction 15 minutes；
2.7 take the fluorescent microsphere that 0.5mg goat-antis rabbit polyclonal antibody (10.0mg/ml takes 50 μ l) is added after above-mentioned activation
In solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
2.8 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
2.9 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.10 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
2.11 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.12 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
3 prepare the fluoroscopic examination card of TIMP-2 in Quantitative detection human blood
3.1 prepare sample pad：Using glass fibre as urine specimen pad, immersion containing 1% skimmed milk power or BSA,
0.1% Tween-20, the phosphate buffers of 0.1M PH 7.40, drying at room temperature, hermetic bag hermetically drying are preserved.
3.2 prepare fluorescence pad：It is mono- that the specific high-affinity restructuring TIMP-2 of mark is diluted respectively with fluorescent microsphere redissolution liquid
The fluorescent microsphere of clonal antibody and the fluorescent microsphere of goat anti-rabbit igg polyclonal antibody, 1 is pressed by the microballoon after mark:1 volume ratio is mixed
Close, be then added on polyester film pad, be freeze-dried, hermetic bag hermetically drying is preserved.
3.3 prepare reacting pad：
It is coated with buffer solution：0.1M PH 8 phosphate, 5wt% sucrose, 0.05wt% Tween-20.Another strain of dilution
Mouse-anti-human T IMP-2 MAb concentrations are 0.8mg/ml, and the rabbit igg concentration of dilution is 1.0mg/ml.
Nitrocellulose filter：Outsourcing
Prepare reacting pad:Diluting another plant of mouse-anti-human T IMP-2 MAb concentration respectively with coating buffer solution is
0.8mg/ml, the rabbit igg concentration of dilution is 1.0mg/ml.Then using dilution after two kinds of antibody on nitrocellulose filter according to
Secondary parallel line forms detection line, nature controlling line.
3.4 blotting papers, bottom plate outsourcing.
3.5 prepare the fluoroscopic examination card of TIMP-2 in Quantitative detection human urine：Will be by sample pad, fluorescence pad, reaction
Pad, blotting paper are combined into as shown in Figure 1 with bottom plate.
Comparative example 7
1st, the preparation of TIMP-2 antibody labelings fluorescent microsphere：
1.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
1.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
1.3 20000 revs/min of centrifugations are centrifuged 30 minutes；
1.4 remove supernatant, and precipitation is resuspended with 1ml PBSs, and makes microballoon within ultrasonically treated 10 minutes with Ultrasound Instrument
Solution is uniformly dispersed；
1.5 add amino-polyethyleneglycols carboxyl, amino-polyethyleneglycols into the fluorescent microsphere solution after above-mentioned centrifugal treating
Carboxyl final concentration 100mM, 37 DEG C are reacted 1~3 hour；
Above-mentioned reacted microspheres solution is loaded bag filter by 1.6, is dialysed in 200ml MES buffer solutions, it is unnecessary to remove
Reaction residue；
1.7 add EDC solution into the fluorescent microsphere solution after above-mentioned dialysis (adds 0.1ml per 1ml fluorescent microsphere liquid
EDC solution), room temperature priming reaction 15 minutes；
1.8 take the fluorescence that 0.6mg mouse-anti-human T IMP-2 monoclonal antibodies (6.9mg/ml takes 87 μ l) are added after above-mentioned activation
In microspheres solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
1.9 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
1.10 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.11 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
1.12 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.13 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
2nd, the preparation of goat anti-rabbit antibody mark fluorescent microballoon：
2.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
2.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
2.3 20000 revs/min of centrifugations are centrifuged 30 minutes；
2.4 remove supernatant, and precipitation is resuspended with 1ml PBSs, and makes microballoon within ultrasonically treated 10 minutes with Ultrasound Instrument
Solution is uniformly dispersed；
2.5 add amino-polyethyleneglycols carboxyl, amino-polyethyleneglycols carboxyl into the fluorescent microsphere solution after above-mentioned centrifugation
Final concentration 50mM, 37 DEG C are reacted 1~3 hour；
Above-mentioned reacted microspheres solution is loaded bag filter by 2.6, is dialysed in 200ml MES buffer solutions, it is unnecessary to remove
Reaction residue；
2.7 add EDC solution into the fluorescent microsphere solution after above-mentioned dialysis (adds 0.1ml per 1ml fluorescent microsphere liquid
EDC solution), room temperature priming reaction 15 minutes；
2.8 take the fluorescent microsphere that 0.5mg goat-antis rabbit polyclonal antibody (10.0mg/ml takes 50 μ l) is added after above-mentioned activation
In solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
2.9 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
2.10 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.11 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
2.12 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.13 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
3.1 prepare the fluoroscopic examination card of TIMP-2 in detection urine：Described in method be the same as Example 4.
Comparative example 8
1st, the preparation of TIMP-2 antibody labelings fluorescent microsphere：
1.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
1.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
Fluorescent microsphere solution after above-mentioned activation is loaded bag filter (molecular cut off 30KD) by 1.3, in 200ml PBS
In 2~8 DEG C of dialysis in buffer solution, unnecessary reaction residue is removed；
1.4 take the fluorescence that 0.6mg mouse-anti-human T IMP-2 monoclonal antibodies (6.5mg/ml takes 87 μ l) are added after above-mentioned activation
In microspheres solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
1.5 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
1.6 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.7 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
1.8 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.9 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
2nd, the preparation of goat anti-rabbit antibody mark fluorescent microballoon：
2.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
2.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
Fluorescent microsphere solution after above-mentioned activation is loaded bag filter (molecular cut off 30KD) by 2.3, in 200ml PBS
In 2~8 DEG C of dialysis in buffer solution, unnecessary reaction residue is removed；
2.4 take the fluorescent microsphere that 0.5mg goat-antis rabbit polyclonal antibody (10.0mg/ml takes 50 μ l) is added after above-mentioned activation
In solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
2.5 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
2.6 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.7 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
2.8 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.9 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
3.1 prepare the fluoroscopic examination card of TIMP-2 in detection human urine：Described in method be the same as Example 4.
Test data：
Embodiment 4, comparative example 7, the test and comparison result of comparative example 8 are as follows：
TIMP-2 immunofluorescence detection agent cards prepared by embodiment 4, comparative example 7, the method for comparative example 8 are respectively adopted,
Difference of the detection reagent on calibration work curve prepared by test and comparison different process method, and sensitivity for analysis,
Difference in range of linearity main performance index：
A uses gradient concentration TIMP-2 calibration objects (1:100 dilution) the calibration results and its working curve as shown in Figure 8
From the calibration results, the calibration results signal value of embodiment 4 is apparently higher than comparing example 7 and comparative example 8；Embodiment
Each concentration point signaling zone is indexed apparently higher than comparative example 7 and comparative example 8. on 4 working curves
B sensitivity for analysis
Compare fluorescence signal between 20ng/ml samples and blank sample (0ng/ml) poor
Δ difference：The same TIMP-2 low concentration samples (20ng/ml) of test, the signal sensitivity of embodiment 4 apparently higher than
Comparative example 7 and comparative example 8
The C ranges of linearity
The TIMP-2 detection reagents card prepared respectively using three kinds of different process methods detects high, medium and low various concentrations model
The concentration known sample enclosed, compares the recurrence difference of measured value and theoretical value.
As shown in figure 9, the regression result is visible, the range of linearity performance of embodiment 4 is substantially better than comparative example 7 and comparative example
8。
Embodiment 5
In embodiment 5 and comparative example 9,10：
Fluorescent microsphere liquid：Particle diameter 300nm, solid content 1%；
EDC solution：50mg/ml 1- (3- dimethylaminopropyls) -3 ethyl carbodiimides (EDC),；
NHS solution：50mg/ml n-hydroxysuccinimides (NHS)；
MES buffer solutions：0.1M MES PH 5N- morpholino b acids (MES),；
PBS：The phosphate (PBS) of 0.05M PBS PH 7.4；
Amino-polyethyleneglycols carboxyl (NH2- PEG-COOH), molecular weight is 2000Dalton.
Fluorescent microsphere redissolves liquid：Containing 0.2wt%BSA, 0.1wt% Qula X-100,0.08wt% Sodium azide, 0.1M, PH
8.0 phosphate buffer.
Prepare the fluoroscopic examination card of IL-18 in Quantitative detection urine
1st, the preparation of IL-18 antibody labelings fluorescent microsphere：
1.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
1.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
Fluorescent microsphere solution after above-mentioned activation is loaded bag filter (molecular cut off 30KD) by 1.3, in 200ml PBS
In 2~8 DEG C of dialysis in buffer solution, unnecessary reaction residue is removed；
1.4 add amino-polyethyleneglycols carboxyl, amino-polyethyleneglycols carboxyl into the fluorescent microsphere solution after above-mentioned dialysis
Final concentration 50mM, 37 DEG C are reacted 1~3 hour；
Above-mentioned reacted microspheres solution is loaded bag filter by 1.5, is dialysed in 200ml MES buffer solutions, it is unnecessary to remove
Reaction residue；
1.6 add EDC solution into the fluorescent microsphere solution after above-mentioned dialysis (adds 0.1ml per 1ml fluorescent microsphere liquid
EDC solution), room temperature priming reaction 15 minutes；
1.7 take the fluorescence that the anti-human IL-18 monoclonal antibodies of 1.2mg mouse (8.0mg/ml takes 182 μ l) are added after above-mentioned activation
In microspheres solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
1.8 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
1.9 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.10 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
1.11 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.12 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
2nd, the preparation of goat anti-rabbit antibody mark fluorescent microballoon：
2.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
2.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
Fluorescent microsphere solution after above-mentioned activation is loaded bag filter (molecular cut off 30KD) by 2.3, in 200ml PBS
In 2~8 DEG C of dialysis in buffer solution, unnecessary reaction residue is removed；
2.4 add amino-polyethyleneglycols carboxyl, amino-polyethyleneglycols carboxyl into the fluorescent microsphere solution after above-mentioned dialysis
Final concentration 50mM, 37 DEG C are reacted 1~3 hour；
Above-mentioned reacted microspheres solution is loaded bag filter by 2.5, is dialysed in 200ml MES buffer solutions, it is unnecessary to remove
Reaction residue；
2.6 add EDC solution into the fluorescent microsphere solution after above-mentioned dialysis (adds 0.1ml per 1ml fluorescent microsphere liquid
EDC solution), room temperature priming reaction 15 minutes；
2.7 take the fluorescent microsphere that 0.5mg goat-antis rabbit polyclonal antibody (10.0mg/ml takes 50 μ l) is added after above-mentioned activation
In solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
2.8 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
2.9 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.10 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
2.11 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.12 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
3 prepare the fluoroscopic examination card of IL-18 in Quantitative detection human urine
3.1 prepare sample pad：Using glass fibre as urine specimen pad, immersion containing 1% skimmed milk power or BSA,
0.1% Tween-20, the phosphate buffers of 0.1M PH 7.40, drying at room temperature, hermetic bag hermetically drying are preserved.
3.2 prepare fluorescence pad：It is anti-human that the specific high-affinity recombinant murine of mark is diluted respectively with fluorescent microsphere redissolution liquid
The fluorescent microsphere of IL-18 monoclonal antibodies and the fluorescent microsphere of goat anti-rabbit igg polyclonal antibody, 1 is pressed by the microballoon after mark:1
Volume ratio is mixed, and is then added on polyester film pad, is freeze-dried, and hermetic bag hermetically drying is preserved.
3.3 prepare reacting pad：
It is coated with buffer solution：0.1M PH 8 phosphate, 5wt% sucrose, 0.05wt% Tween-20.Another strain of dilution
The anti-human IL-18 antibody concentrations of mouse are 0.8mg/ml, and the IgG concentration of dilution is 1.0mg/ml.
Nitrocellulose filter：Outsourcing
Prepare reacting pad:Diluting the anti-human IL-18 monoclonal antibodies of another plant of mouse to concentration respectively with coating buffer solution is
0.8mg/ml, with coating buffer solution dilution rabbit igg, the IgG concentration of dilution is 1.0mg/ml.Then resisted using two kinds after dilution
Body line parallel successively on nitrocellulose filter forms detection line, nature controlling line.
3.4 blotting papers, bottom plate outsourcing.
3.5 prepare the fluoroscopic examination card of IL-18 in Quantitative detection human urine：Will be by sample pad, fluorescence pad, reaction
Pad, blotting paper are combined into as shown in Figure 1 with bottom plate.
Comparative example 9
1st, the preparation of IL-18 antibody labelings fluorescent microsphere：
1.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
1.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
1.3 20000 revs/min of centrifugations are centrifuged 30 minutes；
1.4 remove supernatant, and precipitation is resuspended with 1ml PBSs, and makes microballoon within ultrasonically treated 10 minutes with Ultrasound Instrument
Solution is uniformly dispersed；
1.5 add amino-polyethyleneglycols carboxyl, amino-polyethyleneglycols carboxyl into the fluorescent microsphere solution after above-mentioned centrifugation
Final concentration 50mM, 37 DEG C are reacted 1~3 hour；
Above-mentioned reacted microspheres solution is loaded bag filter by 1.6, is dialysed in 200ml MES buffer solutions, it is unnecessary to remove
Reaction residue；
1.7 add EDC solution into the fluorescent microsphere solution after above-mentioned dialysis (adds 0.1ml per 1ml fluorescent microsphere liquid
EDC solution), room temperature priming reaction 15 minutes；
1.8 take the fluorescent microsphere that 1.2mg IL-18 monoclonal antibodies (6.6mg/ml takes 182 μ l) are added after above-mentioned activation
In solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
1.9 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
1.10 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.11 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
1.12 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.13 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
2nd, the preparation of goat anti-rabbit antibody mark fluorescent microballoon：
2.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
2.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
2.3 20000 revs/min of centrifugations are centrifuged 30 minutes；
2.4 remove supernatant, and precipitation is resuspended with 1ml PBSs, and makes microballoon within ultrasonically treated 10 minutes with Ultrasound Instrument
Solution is uniformly dispersed；
2.5 add amino-polyethyleneglycols carboxyl, amino-polyethyleneglycols carboxyl into the fluorescent microsphere solution after above-mentioned centrifugation
Final concentration 50mM, 37 DEG C are reacted 1~3 hour；
Above-mentioned reacted microspheres solution is loaded bag filter by 2.6, is dialysed in 200ml MES buffer solutions, it is unnecessary to remove
Reaction residue；
2.7 add EDC solution into the fluorescent microsphere solution after above-mentioned dialysis (adds 0.1ml per 1ml fluorescent microsphere liquid
EDC solution), room temperature priming reaction 15 minutes；
2.8 take the fluorescent microsphere that 0.5mg goat-antis rabbit polyclonal antibody (10.0mg/ml takes 50 μ l) is added after above-mentioned activation
In solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
2.9 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
2.10 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.11 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
2.12 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.13 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
3.1 prepare the fluoroscopic examination card of IL-18 in detection urine：Described in method be the same as Example 5.
Comparative example 10
1st, the preparation of IL-18 antibody labelings fluorescent microsphere：
1.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
1.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
Fluorescent microsphere solution after above-mentioned activation is loaded bag filter (molecular cut off 30KD) by 1.3, in 200ml PBS
In 2~8 DEG C of dialysis in buffer solution, unnecessary reaction residue is removed；
1.4 take the fluorescence that the anti-human IL-18 monoclonal antibodies of 1.2mg mouse (6.6mg/ml takes 182 μ l) are added after above-mentioned activation
In microspheres solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
1.5 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
1.6 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.7 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
1.8 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.9 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
2nd, the preparation of goat anti-rabbit antibody mark fluorescent microballoon：
2.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
2.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
Fluorescent microsphere solution after above-mentioned activation is loaded bag filter (molecular cut off 30KD) by 2.3, in 200ml PBS
In 2~8 DEG C of dialysis in buffer solution, unnecessary reaction residue is removed；
2.4 take the fluorescent microsphere that 0.5mg goat-antis rabbit polyclonal antibody (10.0mg/ml takes 50 μ l) is added after above-mentioned activation
In solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
2.5 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
2.6 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.7 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
2.8 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.9 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
3.1 prepare the fluoroscopic examination card of IL-18 in detection human urine：Described in method be the same as Example 5.
Test data：
Embodiment 5, comparative example 9, the test and comparison result of comparative example 10 are as follows：
IL-18 immunofluorescence detection agent cards prepared by embodiment 5, comparative example 9, the method for comparative example 10 are respectively adopted,
Difference of the detection reagent on calibration work curve prepared by test and comparison different process method, and sensitivity for analysis,
Difference in range of linearity main performance index：
A uses the calibration results and its working curve of gradient concentration IL-18 calibration objects as shown in Figure 10
From the calibration results, the calibration results signal value of embodiment 5 is apparently higher than comparing example 9 and comparative example 10；Implement
Each concentration point signaling zone is indexed apparently higher than comparative example 9 and comparative example 10. on the working curve of example 5
B sensitivity for analysis
Compare fluorescence signal between 0.01ng/ml samples and blank sample (0ng/ml) poor
Δ difference：The same IL-18 low concentration samples (0.01ng/ml) of test, the signal sensitivity of embodiment 5 apparently higher than
Comparative example 9 and comparative example 10.
C. the range of linearity
The IL-18 detection reagents card prepared respectively using three kinds of different process methods detects high, medium and low various concentrations scope
Concentration known sample, compare the recurrence difference of measured value and theoretical value.
Regression result as shown in figure 11 is visible, and the range of linearity performance of embodiment 5 is substantially better than comparative example 9 and comparative example
10。
Embodiment 6
In embodiment 6 and comparative example 11,12：
Fluorescent microsphere liquid：Particle diameter 300nm, solid content 1%；
EDC solution：50mg/ml 1- (3- dimethylaminopropyls) -3 ethyl carbodiimides (EDC),；
NHS solution：50mg/ml n-hydroxysuccinimides (NHS)；
MES buffer solutions：0.1M MES PH 5N- morpholino b acids (MES),；
PBS：The phosphate (PBS) of 0.05M PBS PH 7.4；
Amino-polyethyleneglycols carboxyl (NH2- PEG-COOH), molecular weight is 2000Dalton.
Fluorescent microsphere redissolves liquid：Containing 0.2wt%BSA, 0.1wt% Qula X-100,0.08wt% Sodium azide, 0.1M, PH
8.0 phosphate buffer.
Prepare the fluoroscopic examination card of PCT in Quantitative detection blood
1st, the preparation of PCT antibody labelings fluorescent microsphere：
1.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
1.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
Fluorescent microsphere solution after above-mentioned activation is loaded bag filter (molecular cut off 30KD) by 1.3, in 200ml PBS
In 2~8 DEG C of dialysis in buffer solution, unnecessary reaction residue is removed；
1.4 add amino-polyethyleneglycols carboxyl, amino-polyethyleneglycols carboxyl into the fluorescent microsphere solution after above-mentioned dialysis
Final concentration 50mM, 37 DEG C are reacted 1~3 hour；
Above-mentioned reacted microspheres solution is loaded bag filter by 1.5, is dialysed in 200ml MES buffer solutions, it is unnecessary to remove
Reaction residue；
1.6 add EDC solution into the fluorescent microsphere solution after above-mentioned dialysis (adds 0.1ml per 1ml fluorescent microsphere liquid
EDC solution), room temperature priming reaction 15 minutes；
1.7 fluorescent microspheres for taking 0.5mg PCT monoclonal antibodies (3.2mg/ml takes 156 μ l) to add after above-mentioned activation are molten
In liquid, mix immediately, 2~8 DEG C are reacted 8~12 hours；
1.8 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
1.9 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.10 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
1.11 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.12 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
2nd, the preparation of goat anti-rabbit antibody mark fluorescent microballoon：
2.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
2.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
Fluorescent microsphere solution after above-mentioned activation is loaded bag filter (molecular cut off 30KD) by 2.3, in 200ml PBS
In 2~8 DEG C of dialysis in buffer solution, unnecessary reaction residue is removed；
2.4 add amino-polyethyleneglycols carboxyl, amino-polyethyleneglycols carboxyl into the fluorescent microsphere solution after above-mentioned dialysis
Final concentration 50mM, 37 DEG C are reacted 1~3 hour；
Above-mentioned reacted microspheres solution is loaded bag filter by 2.5, is dialysed in 200ml MES buffer solutions, it is unnecessary to remove
Reaction residue；
2.6 add EDC solution into the fluorescent microsphere solution after above-mentioned dialysis (adds 0.1ml per 1ml fluorescent microsphere liquid
EDC solution), room temperature priming reaction 15 minutes；
2.7 take the fluorescent microsphere that 0.5mg goat-antis rabbit polyclonal antibody (10.0mg/ml takes 50 μ l) is added after above-mentioned activation
In solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
2.8 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
2.9 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.10 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
2.11 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.12 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
3 prepare the fluoroscopic examination card of PCT in Quantitative detection human blood
3.1 prepare sample pad：Using glass fibre as blood sample pad, immersion containing 1% skimmed milk power or BSA,
0.1% Tween-20, the phosphate buffers of 0.1MOL/L PH 7.40, drying at room temperature, hermetic bag hermetically drying are preserved.
3.2 prepare fluorescence pad：It is anti-human that the specific high-affinity recombinant murine of mark is diluted respectively with fluorescent microsphere redissolution liquid
The fluorescent microsphere of PCT monoclonal antibodies and the fluorescent microsphere of goat anti-rabbit igg polyclonal antibody, 1 is pressed by the microballoon after mark:1 body
Then product is added on polyester film pad than mixing, is freeze-dried, and hermetic bag hermetically drying is preserved.
3.3 prepare reacting pad：
It is coated with buffer solution：0.1MOL/L PH 8 phosphate, 5wt% sucrose, 0.05wt% Tween-20.
Nitrocellulose filter：Outsourcing
Prepare reacting pad:Diluting the anti-human PCT monoclonal antibodies of another plant of mouse to concentration respectively with coating buffer solution is
1.0mg/ml, is 1.0mg/ml with coating buffer solution dilution rabbit igg to concentration.Then using two kinds of antibody after dilution in nitric acid
Line parallel successively forms detection line, nature controlling line on cellulose membrane.
3.4 blotting papers, bottom plate outsourcing.
3.5 prepare the fluoroscopic examination card of PCT in Quantitative detection human blood：Will by sample pad, fluorescence pad, reacting pad,
Blotting paper and bottom plate are combined into as shown in Figure 1.
Comparative example 11
1st, the preparation of PCT antibody labelings fluorescent microsphere：
1.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
1.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
1.3 20000 revs/min of centrifugations are centrifuged 30 minutes；
1.4 remove supernatant, and precipitation is resuspended with 1ml PBSs, and makes microballoon within ultrasonically treated 10 minutes with Ultrasound Instrument
Solution is uniformly dispersed；
1.5 add amino-polyethyleneglycols carboxyl, amino-polyethyleneglycols into the fluorescent microsphere solution after above-mentioned centrifugal treating
Carboxyl final concentration 50mM, 37 DEG C are reacted 1~3 hour；
Above-mentioned reacted microspheres solution is loaded bag filter by 1.6, is dialysed in 200ml MES buffer solutions, it is unnecessary to remove
Reaction residue；
1.7 add EDC solution into the fluorescent microsphere solution after above-mentioned dialysis (adds 0.1ml per 1ml fluorescent microsphere liquid
EDC solution), room temperature priming reaction 15 minutes；
1.8 fluorescent microspheres for taking 0.5mg PCT monoclonal antibodies (3.2mg/ml takes 156 μ l) to add after above-mentioned activation are molten
In liquid, mix immediately, 2~8 DEG C are reacted 8~12 hours；
1.9 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
1.10 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.11 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
1.12 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.13 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
2nd, the preparation of goat anti-rabbit antibody mark fluorescent microballoon：
2.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
2.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
2.3 20000 revs/min of centrifugations are centrifuged 30 minutes；
2.4 remove supernatant, and precipitation is resuspended with 1ml PBSs, and makes microballoon within ultrasonically treated 10 minutes with Ultrasound Instrument
Solution is uniformly dispersed；
2.5 add amino-polyethyleneglycols carboxyl, amino-polyethyleneglycols carboxyl into the fluorescent microsphere solution after above-mentioned centrifugation
Final concentration 50mM, 37 DEG C are reacted 1~3 hour；
Above-mentioned reacted microspheres solution is loaded bag filter by 2.6, is dialysed in 200ml MES buffer solutions, it is unnecessary to remove
Reaction residue；
2.7 add EDC solution into the fluorescent microsphere solution after above-mentioned dialysis (adds 0.1ml per 1ml fluorescent microsphere liquid
EDC solution), room temperature priming reaction 15 minutes；
2.8 take the fluorescent microsphere that 0.5mg goat-antis rabbit polyclonal antibody (10.0mg/ml takes 50 μ l) is added after above-mentioned activation
In solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
2.9 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
2.10 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.11 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
2.12 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.13 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
3.1 prepare the fluoroscopic examination card of PCT in detection blood：Described in method be the same as Example 6.
Comparative example 12
1st, the preparation of PCT antibody labelings fluorescent microsphere：
1.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
1.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
Fluorescent microsphere solution after above-mentioned activation is loaded bag filter (molecular cut off 30KD) by 1.3, in 200ml PBS
In 2~8 DEG C of dialysis in buffer solution, unnecessary reaction residue is removed；
1.6 fluorescence for taking the anti-human PCT monoclonal antibodies of 0.5mg mouse (3.2mg/ml takes 156 μ l) to add after above-mentioned activation are micro-
In ball solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
1.7 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
1.8 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.9 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
1.10 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.11 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
2nd, the preparation of goat anti-rabbit antibody mark fluorescent microballoon：
2.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
2.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
Fluorescent microsphere solution after above-mentioned activation is loaded bag filter (molecular cut off 30KD) by 2.3, in 200ml PBS
In 2~8 DEG C of dialysis in buffer solution, unnecessary reaction residue is removed；
2.4 take the fluorescent microsphere that 0.5mg goat-antis rabbit polyclonal antibody (10.0mg/ml takes 50 μ l) is added after above-mentioned activation
In solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
2.5 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
2.6 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.7 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
2.8 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.9 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
3.1 prepare the fluoroscopic examination card of PCT in detection human blood：Described in method be the same as Example 6.
Test data：
Embodiment 6, comparative example 11, the test and comparison result of comparative example 12 are as follows：
PCT immunofluorescence detection agent cards prepared by embodiment 6, comparative example 11, the method for comparative example 12 are respectively adopted, survey
Difference of the detection reagent on calibration work curve prepared by different process method is compared in examination, and in sensitivity for analysis, line
Difference in property scope main performance index：
A uses the calibration results and its working curve of gradient concentration PCT calibration objects as shown in Figure 12
From the calibration results, the calibration results signal value of embodiment 6 is apparently higher than comparing example 11 and comparative example 12；Implement
Each concentration point signaling zone is indexed apparently higher than comparative example 11 and comparative example 12. on the working curve of example 6
B sensitivity for analysis
Compare fluorescence signal between 0.1ng/ml samples and blank sample (0ng/ml) poor
Δ difference：The same PCT low concentration samples (0.1ng/ml) of test, the signal sensitivity of embodiment 6 is apparently higher than right
Ratio 11 and comparative example 12.
The C ranges of linearity
The PCT detection reagents card prepared respectively using three kinds of different process methods detects high, medium and low various concentrations scope
Concentration known sample, compares the recurrence difference of measured value and theoretical value.
Regression result as shown in figure 13 is visible, and the range of linearity performance of embodiment 6 is substantially better than comparative example 11 and comparative example
12。
Embodiment 7
In embodiment 7 and comparative example 13,14：
Fluorescent microsphere liquid：Particle diameter 300nm, solid content 1%；
EDC solution：50mg/ml 1- (3- dimethylaminopropyls) -3 ethyl carbodiimides (EDC),；
NHS solution：50mg/ml n-hydroxysuccinimides (NHS)；
MES buffer solutions：0.1M MES PH 5N- morpholino b acids (MES),；
PBS：The phosphate (PBS) of 0.05M PBS PH 7.4；
Amino-polyethyleneglycols carboxyl (NH2- PEG-COOH), molecular weight is 2000Dalton.
Fluorescent microsphere redissolves liquid：Containing 0.2wt%BSA, 0.1wt% Qula X-100,0.08wt% Sodium azide, 0.1M, PH
8.0 phosphate buffer.
Prepare the fluoroscopic examination card of cTnI in Quantitative detection blood
1st, the preparation of cTnI antibody labelings fluorescent microsphere：
1.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
1.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
Fluorescent microsphere solution after above-mentioned activation is loaded bag filter (molecular cut off 30KD) by 1.3, in 200ml PBS
In 2~8 DEG C of dialysis in buffer solution, unnecessary reaction residue is removed；
1.4 add amino-polyethyleneglycols carboxyl, amino-polyethyleneglycols carboxyl into the fluorescent microsphere solution after above-mentioned dialysis
Final concentration 50mM, 37 DEG C are reacted 1~3 hour；
Above-mentioned reacted microspheres solution is loaded bag filter by 1.5, is dialysed in 200ml MES buffer solutions, it is unnecessary to remove
Reaction residue；
1.6 add EDC solution into the fluorescent microsphere solution after above-mentioned dialysis (adds 0.1ml per 1ml fluorescent microsphere liquid
EDC solution), room temperature priming reaction 15 minutes；
1.7 fluorescent microspheres for taking 0.9mg cTNI monoclonal antibodies (7.2mg/ml takes 125 μ l) to add after above-mentioned activation are molten
In liquid, mix immediately, 2~8 DEG C are reacted 8~12 hours；
1.8 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
1.9 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.10 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
1.11 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.12 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
2nd, the preparation of goat anti-rabbit antibody mark fluorescent microballoon：
2.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
2.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
Fluorescent microsphere solution after above-mentioned activation is loaded bag filter (molecular cut off 30KD) by 2.3, in 200ml PBS
In 2~8 DEG C of dialysis in buffer solution, unnecessary reaction residue is removed；
2.4 add amino-polyethyleneglycols carboxyl, amino-polyethyleneglycols carboxyl into the fluorescent microsphere solution after above-mentioned dialysis
Final concentration 50mM, 37 DEG C are reacted 1~3 hour；
Above-mentioned reacted microspheres solution is loaded bag filter by 2.5, is dialysed in 200ml MES buffer solutions, it is unnecessary to remove
Reaction residue；
2.6 add EDC solution into the fluorescent microsphere solution after above-mentioned dialysis (adds 0.1ml per 1ml fluorescent microsphere liquid
EDC solution), room temperature priming reaction 15 minutes；
2.7 take the fluorescent microsphere that 0.5mg goat-antis rabbit polyclonal antibody (10.0mg/ml takes 50 μ l) is added after above-mentioned activation
In solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
2.8 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
2.9 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.10 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
2.11 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.12 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
3 prepare the fluoroscopic examination card of cTnI in Quantitative detection human blood
3.1 prepare sample pad：Using glass fibre as urine specimen pad, immersion containing 1% skimmed milk power or BSA,
0.1% Tween-20,0.1M PH7.40 phosphate buffers, drying at room temperature, hermetic bag hermetically drying are preserved.
3.2 prepare fluorescence pad：It is anti-human that the specific high-affinity recombinant murine of mark is diluted respectively with fluorescent microsphere redissolution liquid
The fluorescent microsphere of cTnI monoclonal antibodies and the fluorescent microsphere of goat anti-rabbit igg polyclonal antibody, 1 is pressed by the microballoon after mark:1 body
Then product is added on polyester film pad than mixing, is freeze-dried, and hermetic bag hermetically drying is preserved.
3.3 prepare reacting pad：
It is coated with buffer solution：0.1M PH 8 phosphate, 5wt% sucrose, 0.05wt% Tween-20.
Nitrocellulose filter：Outsourcing
Prepare reacting pad:Diluting the anti-human cTnI monoclonal antibodies of another plant of mouse to concentration respectively with coating buffer solution is
1.2mg/ml, dilutes rabbit igg, the IgG concentration of dilution is 1.0mg/ml respectively with coating buffer solution.Then using two after dilution
Plant antibody line parallel successively on nitrocellulose filter and form detection line, nature controlling line.
3.4 blotting papers, bottom plate outsourcing.
3.5 prepare the fluoroscopic examination card of cTnI in Quantitative detection human blood：Will be by sample pad, fluorescence pad, reaction
Pad, blotting paper are combined into as shown in Figure 1 with bottom plate.
Comparative example 13
1st, the preparation of cTnI antibody labelings fluorescent microsphere：
1.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
1.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
1.3 20000 revs/min of centrifugations are centrifuged 30 minutes；
1.4 remove supernatant, and precipitation is resuspended with 1ml PBSs, and makes microballoon within ultrasonically treated 10 minutes with Ultrasound Instrument
Solution is uniformly dispersed；
1.5 add amino-polyethyleneglycols carboxyl, amino-polyethyleneglycols into the fluorescent microsphere solution after above-mentioned centrifugal treating
Carboxyl final concentration 50mM, 37 DEG C are reacted 1~3 hour；
Above-mentioned reacted microspheres solution is loaded bag filter by 1.6, is dialysed in 200ml MES buffer solutions, it is unnecessary to remove
Reaction residue；
1.7 add EDC solution into the fluorescent microsphere solution after above-mentioned dialysis (adds 0.1ml per 1ml fluorescent microsphere liquid
EDC solution), room temperature priming reaction 15 minutes；
1.8 fluorescent microspheres for taking 0.9mg cTnI monoclonal antibodies (7.2mg/ml takes 125 μ l) to add after above-mentioned activation are molten
In liquid, mix immediately, 2~8 DEG C are reacted 8~12 hours；
1.9 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
1.10 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.11 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
1.12 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.13 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
2nd, the preparation of goat anti-rabbit antibody mark fluorescent microballoon：
2.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
2.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
2.3 20000 revs/min of centrifugations are centrifuged 30 minutes；
2.4 remove supernatant, and precipitation is resuspended with 1ml PBSs, and makes microballoon within ultrasonically treated 10 minutes with Ultrasound Instrument
Solution is uniformly dispersed；
2.5 add amino-polyethyleneglycols carboxyl, amino-polyethyleneglycols into the fluorescent microsphere solution after above-mentioned centrifugal treating
Carboxyl final concentration 50mM, 37 DEG C are reacted 1~3 hour；
Above-mentioned reacted microspheres solution is loaded bag filter by 2.6, is dialysed in 200ml MES buffer solutions, it is unnecessary to remove
Reaction residue；
2.7 add EDC solution into the fluorescent microsphere solution after above-mentioned dialysis (adds 0.1ml per 1ml fluorescent microsphere liquid
EDC solution), room temperature priming reaction 15 minutes；
2.8 take the fluorescent microsphere that 0.5mg goat-antis rabbit polyclonal antibody (10.0mg/ml takes 50 μ l) is added after above-mentioned activation
In solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
2.9 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
2.10 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.11 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
2.12 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.13 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
3.1 prepare the fluoroscopic examination card of cTNI in detection blood：Described in method be the same as Example 7.
Comparative example 14
1st, the preparation of cTNI antibody labelings fluorescent microsphere：
1.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
1.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
Fluorescent microsphere solution after above-mentioned activation is loaded bag filter (molecular cut off 30KD) by 1.3, in 200ml PBS
In 2~8 DEG C of dialysis in buffer solution, unnecessary reaction residue is removed；
1.4 take the fluorescence that the anti-human cTNI monoclonal antibodies of 0.9mg mouse (7.2mg/ml takes 125 μ l) are added after above-mentioned activation
In microspheres solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
1.5 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
1.6 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.7 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
1.8 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
1.9 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
2nd, the preparation of goat anti-rabbit antibody mark fluorescent microballoon：
2.1 take 1ml fluorescent microsphere liquid, add EDC solution 0.1ml, room temperature priming reaction 15 minutes；
2.2 add NHS solution 0.1ml to above-mentioned solution, and room temperature continues to activate 30 minutes；
Fluorescent microsphere solution after above-mentioned activation is loaded bag filter (molecular cut off 30KD) by 2.3, in 200ml PBS
In 2~8 DEG C of dialysis in buffer solution, unnecessary reaction residue is removed；
2.4 take the fluorescent microsphere that 0.5mg goat-antis rabbit polyclonal antibody (10.0mg/ml takes 50 μ l) is added after above-mentioned activation
In solution, mix immediately, 2~8 DEG C are reacted 8~12 hours；
2.5 state bovine serum albumin(BSA) is added in solution then up, bovine serum albumin(BSA) final concentration 1wt%, 37 DEG C of closings
1 hour；
2.6 by above-mentioned solution centrifugal, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.7 remove supernatant, and precipitation is resuspended with PBS, ultrasonic disperse；
2.8 centrifuge again, and 2~8 DEG C 20000 leaves the heart 15 minutes；
2.9 remove supernatant, and precipitation 50ml fluorescent microspheres redissolve liquid and are resuspended, ultrasonic disperse.
3.1 prepare the fluoroscopic examination card of cTNI in detection human blood：Described in method be the same as Example 7.
Test data：
Embodiment 7, comparative example 13, the test and comparison result of comparative example 14 are as follows：
CTNI immunofluorescence detection agent cards prepared by embodiment 7, comparative example 13, the method for comparative example 14 are respectively adopted,
Difference of the detection reagent on calibration work curve prepared by test and comparison different process method, and sensitivity for analysis,
Difference in range of linearity main performance index：
A uses the calibration results and its working curve of gradient concentration cTNI calibration objects as shown in Figure 14
From the calibration results, the calibration results signal value of embodiment 7 is apparently higher than comparing example 13 and comparative example 14；Implement
Each concentration point signaling zone is indexed apparently higher than comparative example 13 and comparative example 14. on the working curve of example 7
B sensitivity for analysis
Compare fluorescence signal between 0.05ng/ml samples and blank sample (0ng/ml) poor
Δ difference：The same cTnI low concentration samples (0.05ng/ml) of test, the signal sensitivity of embodiment 7 apparently higher than
Comparative example 13 and comparative example 14.
The C ranges of linearity
The cTnI detection reagents card prepared respectively using three kinds of different process methods detects high, medium and low various concentrations scope
Concentration known sample, compare the recurrence difference of measured value and theoretical value.
The regression result is visible as shown in Figure 15, and the range of linearity performance of embodiment 7 is substantially better than comparative example 13 and contrast
Example 14.
Claims (21)
1. a kind of preparation method of the fluorescent microsphere of the specific high-affinity recombinant antibodies of mark, it is characterised in that including following
Step：
S1 is with the ethyl carbodiimide solution of 1- (3- dimethylaminopropyls) -3 and n-hydroxysuccinimide solution respectively to glimmering
Light microballoon liquid is activated, and obtains fluorescent microsphere activator mixture；
Fluorescent microsphere activator mixture in step S1 is put into bag filter by S2, and dialysis removes unnecessary in phosphate buffer
1- (3- dimethylaminopropyls) -3 ethyl carbodiimides and n-hydroxysuccinimide, the fluorescent microsphere after being dialysed
Liquid；
S3 will step S2 dialyse after fluorescent microsphere liquid in add amino-polyethyleneglycols carboxyl, 37 DEG C of stirring reactions 1-3 hours,
Carry out dialysing for second in faintly acid N- morpholino b acid cushioning liquid, remove unnecessary amino-polyethyleneglycols carboxyl, obtain two
Fluorescent microsphere liquid after secondary dialysis；The amino-polyethyleneglycols carboxyl molecular weight is 1000~5000Da；
S4 is sub- by adding 1- (3- dimethylaminopropyls) -3 ethyl carbon two by the fluorescent microsphere liquid after bis- dialysis of step S3
Amine is activated again, is added antibody in being reacted 8-12 hours at 2-8 DEG C, is added bovine serum albumin(BSA) and close, centrifugation, then will
Precipitation is resuspended with phosphate buffer PBS, centrifugation, and redissolving liquid with fluorescent microsphere is resuspended, that is, the high parent of mark specificity is made
With the fluorescent microsphere of power recombinant antibodies；
The fluorescent microsphere redissolves liquid to contain 0.1-1.5wt% gelatin or BSA, 0.01-1wt% triton x-100,0.05-
0.1wt% Sodium azides, 0.01-0.2mol/L pH 6.5-8.5 phosphate buffers.
2. the preparation method of the fluorescent microsphere of specific high-affinity recombinant antibodies, its feature are marked according to claim 1
It is：Fluorescent microsphere particle diameter described in step S1 is 250-350nm, solid content 1%；1- (the 3- dimethylaminos third
Base) -3 ethyl carbodiimide solution concentrations be 10-100mg/ml；The n-hydroxysuccinimide mixed solution concentration is 10-
100mg/ml；The fluorescent microsphere liquid, the ethyl carbodiimide solution of 1- (3- dimethylaminopropyls) -3, N- hydroxysuccinimidyls acyl are sub-
Amine mixed liquor volume ratio is 100：1-2：1-2.
3. the preparation method of the fluorescent microsphere of specific high-affinity recombinant antibodies, its feature are marked according to claim 1
It is：The phosphate buffer that buffer solution described in step S2 and step S4 is 10-100mmol/L pH 7.2-7.6.
4. the preparation method of the fluorescent microsphere of specific high-affinity recombinant antibodies, its feature are marked according to claim 1
It is：Amino-polyethyleneglycols carboxyl final concentration 10-100mmol/L in fluorescent microsphere liquid described in step S3.
5. the preparation method of the fluorescent microsphere of specific high-affinity recombinant antibodies, its feature are marked according to claim 1
It is：The N- morpholine second that faintly acid N- morpholino b acids cushioning liquid described in step S3 is 0.1mol/L pH 4.5-6.1
Sulfonic acid cushioning liquid.
6. the preparation method of the fluorescent microsphere of specific high-affinity recombinant antibodies, its feature are marked according to claim 1
It is：Antibody in the step S4 is the anti-human KIM-1 recombinant monoclonal antibodies of mouse and goat anti-rabbit igg polyclonal antibody.
7. the preparation method of the fluorescent microsphere of specific high-affinity recombinant antibodies, its feature are marked according to claim 1
It is：Antibody in the step S4 is the anti-human NGAL recombinant monoclonal antibodies of mouse and goat anti-rabbit igg polyclonal antibody.
8. the preparation method of the fluorescent microsphere of specific high-affinity recombinant antibodies, its feature are marked according to claim 1
It is：Antibody in the step S4 is the anti-human IGFBP-7 recombinant monoclonal antibodies of mouse and goat anti-rabbit igg polyclonal antibody.
9. the preparation method of the fluorescent microsphere of specific high-affinity recombinant antibodies, its feature are marked according to claim 1
It is：Antibody in the step S4 is mouse-anti-human T IMP-2 recombinant monoclonal antibodies and goat anti-rabbit igg polyclonal antibody.
10. the preparation method of the fluorescent microsphere of specific high-affinity recombinant antibodies, its feature are marked according to claim 1
It is：Antibody in the step S4 is the anti-human IL-18 recombinant monoclonal antibodies of mouse and goat anti-rabbit igg polyclonal antibody.
11. the preparation method of the fluorescent microsphere of specific high-affinity recombinant antibodies, its feature are marked according to claim 1
It is：Antibody in the step S4 is the anti-human PCT recombinant monoclonal antibodies of mouse and goat anti-rabbit igg polyclonal antibody.
12. the preparation method of the fluorescent microsphere of specific high-affinity recombinant antibodies, its feature are marked according to claim 1
It is：Antibody in the step S4 is the anti-human cTnI recombinant monoclonal antibodies of mouse and goat anti-rabbit igg polyclonal antibody.
13. with the fluorescence of the specific high-affinity recombinant antibodies of the obtained mark of any method described in claim 1-12
Microballoon.
14. with fluorescence pad made from the fluorescent microsphere of the specific high-affinity recombinant antibodies of mark described in claim 13.
15. a kind of Quantitative detection KIM-1 detection card, it is characterised in that：The detection card includes setting on bottom plate and bottom plate
There are sample pad, fluorescence pad, reacting pad, blotting paper；
The fluorescence pad is the anti-human KIM-1 of specific high-affinity recombinant murine will to be marked made from method described in claim 6
The fluorescent microsphere of antibody and the fluorescent microsphere of goat anti-rabbit igg antibody are by two kinds of fluorescent microspheres 1:1 mixed in equal amounts, is then added in polyester
On film combination pad, what freeze-drying was made；
The reacting pad include reaction pad body and which is provided with coating the anti-human KIM-1 monoclonal antibodies detection line of another plant of mouse and
It is coated with the nature controlling line of rabbit igg.
16. a kind of Quantitative detection NGAL detection card, it is characterised in that：The detection card includes bottom plate and bottom plate is provided with
Sample pad, fluorescence pad, reacting pad, blotting paper；
The fluorescence pad is made from method described in claim 7 the specificity anti-human NGAL of high-affinity recombinant murine will to be marked to resist
The fluorescent microsphere of body and the fluorescent microsphere of goat anti-rabbit igg antibody are by two kinds of fluorescent microspheres 1:1 mixed in equal amounts, is then added in polyester film
On pad, what freeze-drying was made；
The reacting pad includes reaction pad body and which is provided with the anti-human NGAL recombinant monoclonal antibodies detection of another plant of mouse of coating
Line and the nature controlling line for being coated with rabbit igg.
17. a kind of Quantitative detection IGFBP-7 detection card, it is characterised in that：The detection card is included on bottom plate and bottom plate
Provided with sample pad, fluorescence pad, reacting pad, blotting paper；
The fluorescence pad is the anti-human IGFBP- of specific high-affinity recombinant murine will to be marked made from method described in claim 8
The fluorescent microsphere of 7 antibody and goat anti-rabbit igg antibody is by the fluorescent microsphere 1 in two kinds of fluorescent microsphere solution:1 mixed in equal amounts, then
It is added on polyester film pad, what freeze-drying was made；
The reacting pad includes reaction pad body and which is provided with another plant of anti-human IGFBP-7 of mouse of coating recombinant monoclonal antibodies
Detection line and the nature controlling line for being coated with rabbit igg.
18. a kind of Quantitative detection TIMP-2 detection card, it is characterised in that：The detection card includes setting on bottom plate and bottom plate
There are sample pad, fluorescence pad, reacting pad, blotting paper；
The fluorescence pad is will to require to mark the anti-human TIMP-2 antibody of specific high-affinity recombinant murine described in 9 made from method
Fluorescent microsphere with goat anti-rabbit igg antibody is by the fluorescent microsphere 1 in two kinds of fluorescent microsphere solution:1 mixed in equal amounts, is then added in poly-
On ester film combination pad, what freeze-drying was made；
The reacting pad includes reaction pad body and which is provided with another plant of mouse-anti-human T IMP-2 recombinant monoclonal antibodies inspection of coating
Survey line and the nature controlling line for being coated with rabbit igg.
19. a kind of Quantitative detection IL-18 detection card, it is characterised in that：The detection card includes setting on bottom plate and bottom plate
There are sample pad, fluorescence pad, reacting pad, blotting paper；
The fluorescence pad is the anti-human IL-18 of specific high-affinity recombinant murine will to be marked made from method described in claim 10
The fluorescent microsphere of antibody and goat anti-rabbit igg antibody is by the fluorescent microsphere 1 in two kinds of fluorescent microsphere solution:1 mixed in equal amounts, Ran Houjia
On polyester film pad, what freeze-drying was made；
The reacting pad includes reaction pad body and which is provided with the anti-human IL-18 recombinant monoclonal antibodies detection of another plant of mouse of coating
Line and the nature controlling line for being coated with rabbit igg.
20. a kind of Quantitative detection PCT detection card, it is characterised in that：The detection card includes bottom plate and bottom plate is provided with
Sample pad, fluorescence pad, reacting pad, blotting paper；
The fluorescence pad is made from method described in claim 11 the specificity anti-human PCT of high-affinity recombinant murine will to be marked to resist
The fluorescent microsphere of body and goat anti-rabbit igg antibody is by the fluorescent microsphere 1 in two kinds of fluorescent microsphere solution:1 mixed in equal amounts, is then added in
On polyester film pad, what freeze-drying was made；
The reacting pad includes reaction pad body and which is provided with the coating anti-human PCT recombinant monoclonal antibodies detection line of another plant of mouse
With the nature controlling line for being coated with rabbit igg.
21. a kind of Quantitative detection cTnI detection card, it is characterised in that：The detection card includes bottom plate and bottom plate is provided with
Sample pad, fluorescence pad, reacting pad, blotting paper；
The fluorescence pad is the anti-human cTnI of specific high-affinity recombinant murine will to be marked made from method described in claim 12
The fluorescent microsphere of antibody and goat anti-rabbit igg antibody is by the fluorescent microsphere 1 in two kinds of fluorescent microsphere solution:1 mixed in equal amounts, Ran Houjia
On polyester film pad, what freeze-drying was made；
The reacting pad includes reaction pad body and which is provided with the anti-human cTnI recombinant monoclonal antibodies detection of another plant of mouse of coating
Line and the nature controlling line for being coated with rabbit igg.
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| CN107714304A (en) * | 2017-11-10 | 2018-02-23 | 上海速创诊断产品有限公司 | A kind of paper diaper with urine detection function and preparation method thereof |
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| CN111426825A (en) * | 2020-04-08 | 2020-07-17 | 广州赛哲生物科技股份有限公司 | Preparation method of quantum dot microsphere labeled antibody |
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| CN107271692B (en) | 2019-06-21 |
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Denomination of invention: A fluorescent microsphere labeled with specific high affinity recombinant antibody and its application Granted publication date: 20190621 Pledgee: Nantong Branch of Bank of Nanjing Co.,Ltd. Pledgor: Jiangsu Bohua Pharmaceutical Technology Co.,Ltd. Registration number: Y2024980010861 |