CN107219371A - PCT fluorescent micro-ball immune chromatography detection reagent cards and preparation method thereof - Google Patents
PCT fluorescent micro-ball immune chromatography detection reagent cards and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of PCT fluorescent micro-ball immune chromatographies detection reagent card and preparation method thereof, aim to provide a kind of easy to operate, low concentration and the PCT antigens of high concentration, and the reliable PCT fluorescent micro-ball immune chromatographies detection reagent card of testing result can be detected simultaneously；Linking has sample pad, pad, coated film and absorption pad successively on technical points, including bottom plate, described bottom plate, and described pad is coated with the PCT antibody of two kinds of different-grain diameter fluorescent microsphere marks, the chicken IgY of fluorescent microsphere mark；Described coated film T line positions are coated with anti-PCT antibody, and C line positions are coated with goat-anti chicken IgY；Belong to technical field of biological.
Description
Technical field
The invention discloses one kind detection card, and in particular to a kind of PCT fluorescent micro-ball immune chromatographies detection reagent card, this hair
The bright preparation method for also relating to the PCT fluorescent micro-ball immune chromatography detection reagent cards.
Background technology
Procalcitonin（Procalcitonin, PCT）It is a small molecular protein, molecular weight is about 13kDa, generally by first
The C cells of shape gland are produced.PCT has been counted as the outstanding feature thing with systemic inflammatory and septicemia.
PCT, by CALC-1 gene codes, is the precursor of calcitonin.PCT derives from Preprocalcitonin, and the latter is by 141
Amino acid is constituted, and removes signal peptide（1-25 amino acids）The PCT containing 116 amino acid residues is obtained afterwards, and PCT is by continuous
Cracking, ultimately form three molecules, be N-terminal fragment respectively（N-terminal PCT, 57 amino acid residues）, calcitonin（32 amino
Sour residue）With anticalcium element（21 amino acid residues）.
1993, have been reported that discovery PCT levels have been raised in bacterial system infected patient（1）.It is verified, with inflammation
Related PCT is not produced by parafollicular cells of thyroid gland, but is produced by all parenchymal tissues and differentiated class cell（2-4）.PCT
It is a kind of highly desirable bacterium infection mark, because its concentration in normal person is very low, and viral infection can only make PCT
Concentration is slightly elevated.In addition, the prior diagnostic values of PCT be it concentration and inflammation the order of severity it is closely related.
Except inflammation or septicemia can cause PCT rises outer, operation, Multi-place injury, heat shock, burn and it is cardiogenic not
Gram it can equally cause the rise of PCT concentration sometimes.In addition, multinomial research has proven to PCT water after openheart surgery or heart transplant
The importance of flat monitoring, PCT change can be for discriminating acute rejection and by bacterium or fungus-caused infection.
In normal and healthy individual, its serum-concentration is extremely low, only 10-50pg/mL, and conventional method can't detect.
In systemic bacterial, fungi and parasitic infection, system inflammation reaction syndrome, septicemia, acute and chronic pneumonia, acute pancreas
The blood-serum P CT horizontal abnormalities of the patients such as inflammation, active hepatitis, wound increase, and its concentration ratio normal level is several times greater or even up to ten thousand
Times；It infects in virus, autoimmune disease, and organ transplant excludes the concentration in patients serum such as reaction and do not increased or gently
It is micro- to increase.This phenomenon determines PCT high degree of specificity, therefore available for the antidiastole of such disease, in systemic bacterial
There is very high clinical value in terms of infection and the antidiastole of pyemia auxiliary, Index for diagnosis, observation of curative effect, can be widely used for
ICU wards, hematology, surgery, internal medicine, organ transplant section, Experiment on therapy room etc..Current PCT is wide in hospital and hematological system
General use labelling immunoassay technology is radiommunoassay (RIA) and EIA enzyme immunoassay (EIA), but is existed in actual applications
Following defect：The radionuclide contamination of radiommunoassay；The activity of the enzyme of enzyme linked immunological is easily inactivated, the macromolecular mark of enzyme
Note easily influences the space structure of labeled thing.
Immunochromatography (Lateral Flow Immunoassay, LFIA) technology is the one kind risen both at home and abroad in recent years
Quick diagnosis technology.Special antibody (antigen) for carrier, is first fixed on NC films by LFIA with nitrocellulose filter (NC films)
A certain zone, after sample (urine or serum) is added dropwise in film bar one end, due to capillarity, sample will along the film to
Preceding movement, when being moved to the region for being fixed with antibody (antigen), corresponding antigen (antibody) is sent out with the antibody in sample
Raw specific binding, can make the region show certain color with the dyeing such as immune colloid gold, so as to realize specific immune
Diagnosis.Current LFIA quick detection kits are more to be used as label using collaurum, colored latex particles or fluorescein.Base
The quick detection product developed in colloidal gold-labeled method, have that sensitivity is low, be mainly used in qualitative or sxemiquantitative, batch between
The problems such as differing greatly；Although being made moderate progress based on colored latex particles differences between batches, sensitivity is still relatively low, can only also say
Bright qualitative or sxemiquantitative；Immunochromatography sensitivity based on fluorescein labelling technique is greatly improved, and can quantitatively be detected,
But it is due to contain higher autofluorescent background signal in sample, and stock displacements are smaller, can produce large effect to detection.
Time-resolved fluorescence (Time-resolved Fluorescence, TRF) is a kind of heterotope fluorescent marker, and common
Fluorescence is compared, and big with stock displacements, the features such as fluorescence lifetime is long can effectively avoid the background fluorescence in sample, and
The influence of the veiling glares such as exciting light, therefore there is higher sensitivity and antijamming capability compared to common fluorescence.
Chinese patent ZL 201610031801.3 discloses the kit that a kind of time-resolved fluorescence quantitatively detects PCT
, but the kit prepares to fluorescent microsphere antibody complex and requires high, -80 DEG C of low temperature and needs are lyophilized, and detection sensitivity is low,
Only 0.1ng/mL, and serum, detection time length can only be only detected, it is necessary to 20 minutes.
The content of the invention
In view of the above-mentioned problems, it is an object of the invention to provide one kind is easy to operate, low concentration and height can be detected simultaneously
The PCT antigens of concentration, and the reliable PCT fluorescent micro-ball immune chromatographies detection reagent card of testing result.
Another object of the present invention is to provide the preparation method of above-mentioned detection reagent card.
Therefore, first technical scheme that the present invention is provided is such：
On a kind of PCT fluorescent micro-ball immune chromatographies detection reagent card, including bottom plate, described bottom plate successively linking have sample pad,
Pad, coated film and absorption pad, described pad are coated with the PCT antibody of two kinds of different-grain diameter fluorescent microsphere marks, with
And the chicken IgY of fluorescent microsphere mark；Described coated film T line positions are coated with anti-PCT antibody, and C line positions are coated with goat-anti chicken
IgY。
As present invention further optimization, a kind of above-mentioned PCT fluorescent micro-ball immune chromatographies detection reagent card is described
Two kinds of different-grain diameter fluorescent microspheres are that particle diameter is 100nm fluorescent microspheres and 300nm fluorescent microspheres.
As present invention further optimization, a kind of above-mentioned PCT fluorescent micro-ball immune chromatographies detection reagent card is described
Particle diameter is that the proportion of 100nm fluorescent microspheres and 300nm fluorescent microspheres is 5:1~7:1.
The latter solution that the present invention is provided is：
A kind of preparation method of PCT fluorescent micro-ball immune chromatographies detection reagent card, linking has PVC bottom plates, sample successively on bottom plate
Product pad, pad, nitrocellulose membrane and blotting paper, described pad are obtained successively using following methods：
1）Prepare 100nm fluorescent microspheres and mark anti-PCT antibody
The fluorescent microsphere and anti-PCT antibody of 100nm particle diameters are added in 1mL 0.1M pH6.0 4- hydroxyethyl piperazineethanesulfonic acids,
Both proportions are 1:5~1:20；10mg/mL 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochloride solution is added,
30min, 16000rpm centrifugation 30min are reacted at room temperature, supernatant is removed, adds and preserve liquid, ultrasound is mixed；
2）Prepare 300nm fluorescent microspheres and mark anti-PCT antibody
The fluorescent microsphere and anti-PCT antibody of 300nm particle diameters are added in 1mL 0.1M pH6.0 4- hydroxyethyl piperazineethanesulfonic acids,
Both proportions are 1:10~1:50；1- (3- the dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides for adding 10mg/mL are molten
Liquid, reacts at room temperature 30min, 16000rpm centrifugation 30min, removes supernatant, add and preserve liquid, ultrasound is mixed；
3）Prepare fluorescent microsphere mark chicken IgY antibody
Fluorescent microsphere and chicken IgY are added in 1mL 0.1M pH6.0 4- hydroxyethyl piperazineethanesulfonic acids, both proportions are 1:5~
1:20；10mg/mL 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochloride solution is added, 30min is reacted at room temperature.
16000rpm centrifuges 30min, removes supernatant, adds and preserves liquid, and ultrasound is mixed；
4）By above-mentioned steps 1）With step 2）Prepared 100nm, 300nm particle diameter fluorescent microsphere is by weight 5:1~7:1 mixing,
Supernatant is removed in centrifugation after mixing, with 0.1M 3- (N- the morpholinyls) -2- hydroxyls that with the addition of 0.1%SDS, 1%BSA and 0.5%PEG20000
Base propane sulfonic acid redissolves, and obtains mixed mark liquid, mixed marking fluid and step 3）Fluorescent microsphere marks chicken IgY antibody according to weight
Than 20:1 mixing, draws film instrument with metal spraying and is sprayed onto on polyester cellulose film, discharge rate is 3 μ L/cm, is put into 37 DEG C of baking ovens, dries 8 small
When.
Further, a kind of preparation method of above-mentioned PCT fluorescent micro-ball immune chromatographies detection reagent card, described coating
It is anti-PCT antibody that the preparation method of film, which is that T line positions are coated on reaction film, and coated C line positions are goat-anti chicken IgY.
Further, a kind of preparation method of above-mentioned PCT fluorescent micro-ball immune chromatographies detection reagent card, described sample
Sample pad is cut into 20mm × 300mm size by the preparation method of pad, is immersed in sample pad buffer solution, is taken out after 1 hour,
In drying at room temperature 16-18 hours；Described sample pad buffer formulation is as follows：2% sorbose, 0.5% S9 and 0.1mg/mL rabbit-antis
Human red blood cells antibody is dissolved in 0.2M PB pH7.4 buffer solutions.
Further, a kind of preparation method of above-mentioned PCT fluorescent micro-ball immune chromatographies detection reagent card, described fluorescence
The solid content 1% of microballoon.
Further, a kind of preparation method of above-mentioned PCT fluorescent micro-ball immune chromatographies detection reagent card, described preservation
Liquid is：10% sucrose, 1.5%BSA are dissolved in 0.1M Gly-NaOH solution.
Compared with prior art, the PCT fluorescent micro-ball immune chromatography detection reagent fixtures that the present invention is provided have the following advantages：
By the sample after dilution when the technical scheme that the present invention is provided specifically is detected（Serum, blood plasma, whole blood）When adding sample pad,
Sample passes through sample pad successively, and pad will be moved in capillarity sample along reagent strip to the direction of absorption pad.Work as sample
When containing high concentration PCT in product, the PCT in sample can be simultaneously with being coupled two kinds of particle diameters（100nm and 300nm）Fluorescent microsphere it is anti-
With anti-PCT monoclonal antibodies on NC films are coated on specific bond occurs for PCT monoclonal antibodies, in T1Place forms double-antibody sandwich knot
Structure, now the fluorescence intensity at T lines is the summation of two kinds of particle diameter fluorescent microspheres.
When the concentration of PCT in sample is low, the anti-PCT of PCT mainly with the fluorescent microsphere of coupling 300nm particle diameters in sample
With anti-PCT monoclonal antibodies on NC films are coated on specific bond occurs for monoclonal antibody, and double-antibody sandwich structure is formed at T,
Now the fluorescence intensity at T lines is the fluorescence of 300nm particle diameter fluorescent microspheres；Efficiently solving the small fluorescent microsphere of particle diameter can examine
Measure the PCT antigens of high concentration and can not be detected in low concentration, the big fluorescent microsphere of particle diameter can detect the antigen of low concentration,
And go out in high concentration and can not detect, two kinds of particle diameter microballoons of selection of technical scheme that the present invention is provided can be while detect low concentration
With the PCT antigens of high concentration, detection sensitivity height.
Brief description of the drawings
Fig. 1 is the PCT fluorescent micro-ball immune chromatography detection reagent card structure schematic diagrames that the present invention is provided.
Fig. 2 is the PCT fluorescent micro-ball immune chromatography detection reagent card detection curve figures that the present invention is provided.
Embodiment
It is further to the claim of the present invention to be limited with reference to embodiment, but do not constitute to this
Any limitation of invention.
Embodiment 1
A kind of PCT fluorescent micro-ball immune chromatographies detection reagent card that the present invention is provided, refering to Fig. 1, including bottom plate 1, described bottom
Linking has sample pad 2, pad 3, coated film 4 and absorption pad 5 successively on plate 1, and described pad 3 is coated with particle diameter and is
The PCT antibody of 100nm fluorescent microspheres mark and the PCT antibody of 300nm fluorescent microspheres mark, and the chicken that fluorescent microsphere is marked
IgY；Described particle diameter is that the proportion of 100nm fluorescent microspheres and 300nm fluorescent microspheres is 5:1~7:1；Described coated film T lines position
Put and be coated with anti-PCT antibody, C line positions are coated with goat-anti chicken IgY.
The preparation method of above-mentioned PCT fluorescent micro-ball immune chromatography detection reagent cards：
（1）The preparation method of sample pad
The buffer formulation of sample pad is as follows：2% sorbose, 0.5% S9 and 0.1mg/mL rabbit-anti human red blood cells antibody are dissolved in
0.2M PB（pH7.4）In buffer solution.
（2）The preparation method of pad
The step of 100nm fluorescent microspheres mark anti-PCT antibody
The fluorescent microsphere of 100nm particle diameters is added in 1mL 0.1M pH6.0 4- hydroxyethyl piperazineethanesulfonic acids（Solid content 1%）With
Anti- PCT antibody, both proportions are 1:5~1:20；Add 10mg/mL 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides
Hydrochloride（EDC）Solution, reacts at room temperature 30min, 16000rpm centrifugation 30min, removes supernatant, add and preserve liquid, ultrasound is mixed.
The step of 300nm fluorescent microspheres mark anti-PCT antibody
The fluorescent microsphere of 300nm particle diameters is added in 1mL 0.1M pH6.0 4- hydroxyethyl piperazineethanesulfonic acids（Solid content 1%）With
Anti- PCT antibody, both proportions are 1:10~1:50；Add 10mg/mL 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides
Hydrochloride（EDC）Solution, reacts at room temperature 30min.16000rpm centrifuges 30min, removes supernatant, adds and preserves liquid, and ultrasound is mixed.
The step of described fluorescent microsphere mark chicken IgY antibody is：
Fluorescent microsphere is added in 1mL 0.1M pH6.0 4- hydroxyethyl piperazineethanesulfonic acids（Solid content 1%）With chicken IgY, both
Proportion is 1:5~1:20；Add 10mg/mL 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides（EDC）It is molten
Liquid, reacts at room temperature 30min.16000rpm centrifuges 30min, removes supernatant, adds and preserves liquid, and ultrasound is mixed.
Described preservation liquid is：10% sucrose, 1.5%BSA are dissolved in 0.1M Gly-NaOH solution.
By prepared 100nm, 300nm particle diameter fluorescent microsphere by weight 5:1~7:1 mixing（It is preferred that 6:1）, after mixing
Supernatant is removed in centrifugation, with 0.1M 3- (N- morpholinyls) third sulphur of -2- hydroxyls that with the addition of 0.1%SDS, 1%BSA and 0.5%PEG20000
Acid redissolves, and obtains mixed mark liquid, mixed marking fluid and step 3）Fluorescent microsphere mark chicken IgY antibody compares 20 according to weight:1
Mixing, draws film instrument with metal spraying and is sprayed onto on polyester cellulose film, discharge rate is 3 μ L/cm, is put into 37 DEG C of baking ovens, is dried 8 hours.
（3）The preparation method of coated film is：
In detection line T lines by 0.01M PBS（PH7.4 contains 1% glucose）Anti- PCT antibody is diluted to 1mg/mL, picture is carried out
Film, picture film parameters are 1 μ L/cm.
In detection line C lines by 0.01M PBS（PH7.4 contains 1% glucose）Goat-anti chicken IgY antibody is diluted to 1mg/
ML, carries out picture film, and picture film parameters are 1 μ L/cm, then in 37 DEG C of oven dryings, drying time 16h.
The assemble method of above-mentioned PCT fluorescent micro-ball immune chromatography detection reagent cards is as follows：
Each component putting in order on bottom plate is followed successively by absorption pad -- nitrocellulose filter -- pad -- sample pad
1. the stickup of absorption pad：Bottom plate is laid on work top；The diaphragm of bottom plate upper limb absorption pad location for paste is opened, will
Absorption pad is adhered thereto, and uniform, slight roller is promoted, and to strengthen bonding force, and prevents bubble, absorption pad is covered in
2mm on nitrocellulose filter.
2. pad is pasted：It is width 15mm × long 300mm that pad, which is cut out, opens nitrocellulose filter lower edge pad and glues
Diaphragm at patch, pad is adhered thereto, and the same absorption pad of method, pad is covered in 2mm on nitrocellulose filter.
3. the stickup of sample pad：Sample pad is adhered into pad bottom, the same absorption pad of method.Sample pad is covered in combination
2mm on pad.
4. test strips are cut：The bottom plate pasted is put into cutting machine, the wide test strips of 4mm are cut into.
5. it is loaded and enters bag：Each test strips are loaded in plastic clip, each reagent is placed in aluminum foil bag, and is added
1g drier 1 is wrapped, heat sealing.
The specifically used method of PCT fluorescent micro-ball immune chromatography detection reagent cards：
Open instrument, insertion and reagent lot identical chip；Reagent card outer packing is removed when using, reagent card, level is taken out
Place；It is accurate to draw in 75 μ L serum/plasmas after dilution, or 100 μ L whole blood samples, the well for being added to detection card, then
Start timing；After after room temperature reaction 10min, detection card is put into the neck of instrument；" test " button on instrument is clicked on,
Instrument will start test, and show result；Click on " printing ", can print test results report.
Type PCT of the present invention Cleaning Principle is as follows：
By sample（Serum, blood plasma, whole blood）When adding sample well, sample passes through sample pad, pad, in capillarity successively
Sample will be moved along reagent strip to the direction of absorption pad.When containing high concentration PCT in sample, the PCT in sample can be simultaneously
With two kinds of particle diameters of coupling（100nm and 300nm）The anti-PCT monoclonal antibodies of fluorescent microsphere and it is coated on anti-PCT Dan Ke on NC films
Specific bond occurs for grand antibody, and double-antibody sandwich structure is formed at T.Now the fluorescence intensity at T lines is that two kinds of particle diameters are glimmering
The summation of light microballoon.
When the concentration of PCT in sample is low, the anti-PCT of PCT mainly with the fluorescent microsphere of coupling 300nm particle diameters in sample
With anti-PCT monoclonal antibodies on NC films are coated on specific bond occurs for monoclonal antibody, and double-antibody sandwich structure is formed at T,
Now the fluorescence intensity at T lines is the fluorescence of 300nm particle diameter fluorescent microspheres.
So effectively prevent can detect the PCT antigens of high concentration in the small fluorescent microsphere of particle diameter and in low concentration without
Method detects that the big fluorescent microsphere of particle diameter can detect the antigen of low concentration, and go out and can not detect in high concentration.Select certain proportion
Two kinds of particle diameter microballoons can detect low concentration and the PCT antigens of high concentration simultaneously.
In order to prove the effect for the technical scheme that the present invention is provided, following test examples are by PCT calibration objects, and diluted concentration is extremely
0.05ng/mL, 0.5ng/mL, 5ng/mL, 50ng/mL, 100ng/mL sample, take 75 μ L sample to add and add using pipettor
In sample hole, readings in fluorescence detector is put into after standing 10 minutes.Each concentration of specimens is detected three times, with sample after averaging
Concentration value is mapped to detected value, refering to Fig. 2.
| Standard items | 0ng/mL | 0.05ng/mL | 0.5ng/mL | 5ng/mL | 50ng/mL | 100ng/mL |
| Detect T/C values | 0.025 | 0.045 | 0.17 | 1.02 | 10.3 | 19.8 |
In order to better illustrate the beneficial effect of type of the present invention, be given below using type of the present invention provide detection reagent and
Comparative result experiment of traditional enzyme-linked immunosorbent assay, colloidal gold method, fluorescence immune chromatography method when detecting PCT.
| Detection method | Patent publication No. | Test limit |
| Chemiluminescence | CN 106093416 A | 0.05ng/mL |
| Colloidal gold immunochromatographimethod | CN 106093431 A | 0.1ng/mL |
| Latex enhancing immune is than turbid | CN 102759631 B | 0.2ng/mL |
| Fluorescence immune chromatography | CN 104714033 B | 0.1ng/mL |
| The method that the present invention is provided | 0.05ng/mL |
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (8)
1. a kind of PCT fluorescent micro-ball immune chromatographies detection reagent card, including bottom plate（1）, described bottom plate（1）On successively linking have
Sample pad（2）, pad（3）, coated film（4）And absorption pad（5）, it is characterised in that described pad（3）It is coated with two kinds
The PCT antibody of different-grain diameter fluorescent microsphere mark, and the chicken IgY that fluorescent microsphere is marked；Described coated film（4）T line positions
Anti- PCT antibody is coated with, C line positions are coated with goat-anti chicken IgY.
2. a kind of PCT fluorescent micro-ball immune chromatographies detection reagent card according to claim 1, it is characterised in that described
Two kinds of different-grain diameter fluorescent microspheres are that particle diameter is 100nm fluorescent microspheres and 300nm fluorescent microspheres.
3. a kind of PCT fluorescent micro-ball immune chromatographies detection reagent card according to claim 1 or 2, it is characterised in that described
Particle diameter be that the proportion of 100nm fluorescent microspheres and 300nm fluorescent microspheres is 5:1~7:1.
4. a kind of method of PCT fluorescent micro-ball immune chromatographies detection reagent card described in claim is prepared, on bottom plate successively
Linking has PVC bottom plates, sample pad, pad, nitrocellulose membrane and blotting paper, it is characterised in that under described pad is used
State method obtained successively：
1）Prepare 100nm fluorescent microspheres and mark anti-PCT antibody
The fluorescent microsphere and anti-PCT antibody of 100nm particle diameters are added in 1mL 0.1M pH6.0 4- hydroxyethyl piperazineethanesulfonic acids,
Both proportions are 1:5~1:20；10mg/mL 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochloride solution is added,
30min, 16000rpm centrifugation 30min are reacted at room temperature, supernatant is removed, adds and preserve liquid, ultrasound is mixed；
Prepare 300nm fluorescent microspheres and mark anti-PCT antibody
The fluorescent microsphere and anti-PCT antibody of 300nm particle diameters are added in 1mL 0.1M pH6.0 4- hydroxyethyl piperazineethanesulfonic acids,
Both proportions are 1:10~1:50；1- (3- the dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides for adding 10mg/mL are molten
Liquid, reacts at room temperature 30min, 16000rpm centrifugation 30min, removes supernatant, add and preserve liquid, ultrasound is mixed；
Prepare fluorescent microsphere mark chicken IgY antibody
Fluorescent microsphere and chicken IgY are added in 1mL 0.1M pH6.0 4- hydroxyethyl piperazineethanesulfonic acids, both proportions are 1:5~
1:20；10mg/mL 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochloride solution is added, 30min is reacted at room temperature,
16000rpm centrifuges 30min, removes supernatant, adds and preserves liquid, and ultrasound is mixed；
By above-mentioned steps 1）With step 2）Prepared 100nm, 300nm particle diameter fluorescent microsphere is by weight 5:1~7:1 mixing, is mixed
Supernatant is removed in centrifugation after conjunction, with 0.1M 3- (N- the morpholinyls) -2- hydroxyls that with the addition of 0.1%SDS, 1%BSA and 0.5%PEG20000
Propane sulfonic acid redissolves, and obtains mixed mark liquid, mixed marking fluid and step 3）Fluorescent microsphere marks chicken IgY antibody according to weight ratio
20:1 mixing, draws film instrument with metal spraying and is sprayed onto on polyester cellulose film, discharge rate is 3 μ L/cm, is put into 37 DEG C of baking ovens, is dried 8 hours.
5. a kind of preparation method of PCT fluorescent micro-ball immune chromatographies detection reagent card according to claim 4, its feature exists
In it is anti-PCT antibody that the preparation method of described coated film, which is that T line positions are coated on reaction film, and coated C line positions are sheep
Anti- chicken IgY.
6. a kind of preparation method of PCT fluorescent micro-ball immune chromatographies detection reagent card according to claim 4, its feature exists
In sample pad is cut into 20mm × 300mm size by the preparation method of described sample pad, is immersed in sample pad buffer solution, 1
Taken out after hour, in drying at room temperature 16-18 hours；Described sample pad buffer formulation is as follows：2%L- sorboses, 0.5%
S9 and 0.1mg/mL rabbit-anti human red blood cells antibody is dissolved in 0.2M PB pH7.4 buffer solutions.
7. a kind of preparation method of PCT fluorescent micro-ball immune chromatographies detection reagent card according to claim 4, its feature exists
In the solid content 1% of described fluorescent microsphere.
8. a kind of preparation method of PCT fluorescent micro-ball immune chromatographies detection reagent card according to claim 4, its feature exists
In described preservation liquid is：10% sucrose, 1.5%BSA are dissolved in 0.1M Gly-NaOH solution.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108318685A (en) * | 2018-05-04 | 2018-07-24 | 广州敏捷生物技术有限公司 | Immunofluorescence for detecting canine coronavirus antigen chromatographs detection card and preparation method |
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| CN108398565A (en) * | 2018-05-04 | 2018-08-14 | 广州敏捷生物技术有限公司 | Immunofluorescence for detecting dog C reactive protein chromatographs detection card and preparation method |
| CN108414753A (en) * | 2018-05-04 | 2018-08-17 | 广州敏捷生物技术有限公司 | Immunofluorescence for detecting Canine Parvovirus antigen chromatographs detection card and preparation method |
| CN109061200A (en) * | 2018-08-23 | 2018-12-21 | 上海复星长征医学科学有限公司 | Gastrin 17 time resolution micro-ball immune chromatography detection reagent card and preparation method thereof |
| CN109061200B (en) * | 2018-08-23 | 2021-08-06 | 复星诊断科技(上海)有限公司 | Gastrin 17 time-resolved microsphere immunochromatography detection reagent card and preparation method thereof |
| CN110618268A (en) * | 2019-05-10 | 2019-12-27 | 武汉优恩生物科技有限公司 | Method and device for processing fluorescent immunochromatographic test strip binding pad |
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| CN113933519A (en) * | 2021-10-20 | 2022-01-14 | 上海艾瑞德生物科技有限公司 | Test strip and kit for joint detection of CRP and SAA and preparation method |
| CN113933519B (en) * | 2021-10-20 | 2024-06-14 | 上海艾瑞德生物科技有限公司 | Test strip and kit for combined detection of CRP and SAA and preparation method |
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