CN109085363A - Detection reagent, detection kit and the application and detection method of AKAP4 antigen detection - Google Patents
Detection reagent, detection kit and the application and detection method of AKAP4 antigen detection Download PDFInfo
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- CN109085363A CN109085363A CN201810831974.2A CN201810831974A CN109085363A CN 109085363 A CN109085363 A CN 109085363A CN 201810831974 A CN201810831974 A CN 201810831974A CN 109085363 A CN109085363 A CN 109085363A
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
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Abstract
The present invention provides detection reagent, detection kit and the applications and detection method of the detection of AKAP4 antigen, belong to biomedical inspection field.The detection reagent of AKAP4 antigen detection provided by the invention, detection reagent is simple and easy to get, facilitates preparation；It will test reagent to be applied to prepare the detection kit of AKAP4 antigen detection, facilitate and detected；The detection method of AKAP4 antigen detection is easy to operate, and step is simple；It is easily operated；Testing result is accurate and reliable, has preferable practicability.
Description
Technical field
The present invention relates to biomedical inspection fields, detection reagent, detection in particular to the detection of AKAP4 antigen
Kit and application and detection method.
Background technique
Flow cytometry can be used for detecting by the cell of fluorescent marker, embryo, RNA, DNA, protein particulate, micro- life
The biological particles such as object, virus.In detection, flow cytometry needs are radiated at by means of the laser facula Jing Guo shaping positioned at liquid stream
On the biological particle at center, biological particle issues scattering light and fluorescence under the excitation of laser facula.These scattering light and fluorescence
Signal is collected by electrooptical devices such as photomultiplier tube, photodiodes, is analyzed using detection system, to obtain biology
The physics and chemical information of particle, such as cell size, surface granularity, antigenic information.Testing principle based on flow cytometry,
The shape and quality of laser facula are to influence detection sensitivity, the key factor of single particle resolving power.
Current existing survey AKAP4 antigen detection method is enzyme linked immunosorbent assay, the absorbance obtained by microplate reader
Value further calculates its concentration.This method is complicated for operation, and detection needs to establish calibration curve by gradient calibration object every time, no
Conducive to a small amount of pattern detection.And testing result accuracy and calibration object, microplate reader model, precision etc. are in close relations.Therefore,
It is badly in need of one simple, accurate, easy-operating detection method of exploitation.
Summary of the invention
The first object of the present invention is to provide the detection reagent of AKAP4 antigen detection, which is conveniently used for
The detection of AKAP4 antigen.
The second object of the present invention is the detection reagent for providing above-mentioned AKAP4 antigen detection in preparation AKAP4 antigen
It is applied in the detection kit of detection.
The third object of the present invention is to provide the detection kit of AKAP4 antigen detection, is convenient to detect.
The fourth object of the present invention is to provide the detection method of AKAP4 antigen detection, easy to operate, quickly easy.
In order to realize above-mentioned purpose of the invention, using following technical scheme:
The detection reagent of AKAP4 antigen detection, detection reagent mainly includes PBS sample cleaning solution, sample fixer, goat-anti
The antibody of people AKAP4, the secondary antibody of fluorescein-labeled anti-sheep.
The detection reagent of above-mentioned AKAP4 antigen detection is applied in the detection kit for preparing the detection of AKAP4 antigen.
The detection kit of AKAP4 antigen detection, detection kit includes PBS sample cleaning solution, sample fixer, goat-anti
The antibody of people AKAP4, the secondary antibody of fluorescein-labeled anti-sheep, sample fixer be include 0.3% paraformaldehyde PBS buffering
Liquid.
The detection method of AKAP4 antigen detection, detection method includes the following steps:
Cell sample is washed with PBS sample cleaning solution, the fixed cell sample of sample fixer of 1-3 times of volume is added, obtains
To immobilization sample, immobilization sample is divided into detection fixed sample and control fixed sample；
The antibody of 1-100 μ L goat-anti people AKAP4 is added in detection fixed sample, obtains mixing sample to be checked；Control is solid
Non-specific IgG is originally added in random sample, obtains control mixing sample；
Mixing sample to be checked and control mixing sample carry out the first incubation respectively, are then washed respectively with PBS sample cleaning solution
It washs, the secondary antibody that fluorescein-labeled anti-sheep is added carries out second and is incubated for, and then uses PBS sample cleaning solution；
Pass through flow cytomery.
The invention has the benefit that the detection reagent of AKAP4 antigen detection provided by the invention, detection reagent are simply easy
, facilitate preparation；It will test reagent to be applied to prepare the detection kit of AKAP4 antigen detection, facilitate and detected；AKAP4
The detection method of antigen detection is easy to operate, and step is simple；It is easily operated；Testing result is accurate and reliable, has preferable practical
Property.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 is the AKAP4 antigen testing result figure that experimental example of the present invention provides.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Detection reagent, detection kit and application to the AKAP4 antigen detection of the embodiment of the present invention and detection side below
Method is specifically described.
The detection reagent of AKAP4 antigen detection, detection reagent mainly includes PBS sample cleaning solution, sample fixer, goat-anti
The antibody of people AKAP4, the secondary antibody of fluorescein-labeled anti-sheep.
By antigen-antibody reaction, and the fluorescein on antibody is marked, can preferably detected in cell
AKAP4 antigen.
Further, in preferred embodiments of the present invention, sample fixer is the paraformaldehyde for including 0.1%-0.5%
PBS buffer solution.
Further, in preferred embodiments of the present invention, the pH value of PBS sample cleaning solution and sample fixer is 6.0-
8.0。
Further, in preferred embodiments of the present invention, one of fluorescein FITC, PE, PI and Percp.
The use of fluorescein, it is convenient after antigen-antibody reaction occurs, by the detection of fluorescein, further obtain
The information of AKAP4 antigen.
The detection reagent of above-mentioned AKAP4 antigen detection is applied in the detection kit for preparing the detection of AKAP4 antigen.
The detection kit of AKAP4 antigen detection, detection kit includes PBS sample cleaning solution, sample fixer, goat-anti
The antibody of people AKAP4, the secondary antibody of fluorescein-labeled anti-sheep, sample fixer be include 0.3% paraformaldehyde PBS buffering
Liquid.
The detection method of AKAP4 antigen detection, detection method includes the following steps:
Cell sample is washed with PBS sample cleaning solution, the fixed cell sample of sample fixer of 1-3 times of volume is added, obtains
To immobilization sample, immobilization sample is divided into detection fixed sample and control fixed sample；
The antibody of 1-100 μ L goat-anti people AKAP4 is added in detection fixed sample, obtains mixing sample to be checked；Control is solid
Non-specific IgG is originally added in random sample, obtains control mixing sample；
Mixing sample to be checked and control mixing sample carry out the first incubation respectively, are then washed respectively with PBS sample cleaning solution
It washs, the secondary antibody that fluorescein-labeled anti-sheep is added carries out second and is incubated for, and then uses PBS sample cleaning solution；
Pass through flow cytomery.
Further, in preferred embodiments of the present invention, cell sample contains 1 × 104-1×106A/mL cell.
Further, in preferred embodiments of the present invention, the temperature of fixed cell sample is 2-8 DEG C, time 4-
9min。
Further, in preferred embodiments of the present invention, the first temperature being incubated for is 2-8 DEG C, time 10min-
24h；Second temperature being incubated for is 2-8 DEG C, time 5-30min.
Feature and performance of the invention are described in further detail with reference to embodiments.
Embodiment 1
The present embodiment provides the detection reagent of AKAP4 antigen detection, which can be used for detecting in cell sample
AKAP4 antigen.
The detection reagent of AKAP4 antigen detection include PBS sample cleaning solution, sample fixer, goat-anti people AKAP4 it is anti-
Body, the secondary antibody of the anti-sheep of fluorescein FITC label.
Sample fixer be include 0.1% paraformaldehyde PBS buffer solution.PBS sample cleaning solution and sample fixer
PH value be 6.0.
The detection reagent of AKAP4 antigen detection provided in this embodiment can be used for preparing the detection of AKAP4 antigen detection
Kit.
The present embodiment also provides a kind of detection kit of AKAP4 antigen detection, which includes above-mentioned
The detection reagent of AKAP4 antigen detection.
Embodiment 2
The present embodiment provides the detection reagent of AKAP4 antigen detection, which can be used for detecting in cell sample
AKAP4 antigen.
The detection reagent of AKAP4 antigen detection include PBS sample cleaning solution, sample fixer, goat-anti people AKAP4 it is anti-
Body, the secondary antibody of the anti-sheep of fluorescein PE label.
Certain fluorescein can also be one of FITC, PI and Percp, can be with other kinds of fluorescein.
Sample fixer be include 0.3% paraformaldehyde PBS buffer solution.PBS sample cleaning solution and sample fixer
PH value be 8.0.
The detection reagent of AKAP4 antigen detection provided in this embodiment can be used for preparing the detection of AKAP4 antigen detection
Kit.
The present embodiment also provides a kind of detection kit of AKAP4 antigen detection, which includes above-mentioned
The detection reagent of AKAP4 antigen detection.
Embodiment 3
The present embodiment provides the detection reagent of AKAP4 antigen detection, which can be used for detecting in cell sample
AKAP4 antigen.
The detection reagent of AKAP4 antigen detection include PBS sample cleaning solution, sample fixer, goat-anti people AKAP4 it is anti-
Body, the secondary antibody of the anti-sheep of fluorescein PE label.
Certain fluorescein can also be one of FITC, PI and Percp, can be with other kinds of fluorescein.
Sample fixer be include 0.5% paraformaldehyde PBS buffer solution.PBS sample cleaning solution and sample fixer
PH value be 7.0.
The detection reagent of AKAP4 antigen detection provided in this embodiment can be used for preparing the detection of AKAP4 antigen detection
Kit.
The present embodiment also provides a kind of detection kit of AKAP4 antigen detection, which includes above-mentioned
The detection reagent of AKAP4 antigen detection.
Embodiment 4
The present embodiment also provides the detection method of AKAP4 antigen detection, the detection reagent that the present embodiment is provided with embodiment 3
It is detected, detection method includes the following steps for this；
1.1 take cell concentration be 1 × 104The cell sample of a/mL is washed 1-3 times with PBS sample cleaning solution；
1.2 fix washed cell sample with 1 times of sample fixer again, and 2 DEG C of fixed temperature, time 4min is obtained
Immobilization sample；And immobilization sample is divided into detection fixed sample and control fixed sample；
The antibody for the goat-anti people AKAP4 that 1 μ L is added in 1.3 detection fixed samples obtains mixing sample to be checked, compares fixed sample
The non-specific IgG of this addition equivalent obtains control mixing sample；
1.4 mixing samples to be checked and control mixing sample, carry out first respectively under the conditions of 2 DEG C and are incubated for for 24 hours；
The mixing sample to be checked and control mixing sample being incubated in 1.5 steps 1.4 are washed with PBS sample cleaning solution respectively
1-3 times；Then the secondary antibody of the anti-sheep of fluorescein PE label is added；Second is carried out under the conditions of 2 DEG C respectively is incubated for 30min；
1.6 mixing samples to be checked being incubated for through second and control mixing sample are washed 1-3 times with PBS sample cleaning solution, so
After pass through flow cytomery.
Embodiment 5
The present embodiment also provides the detection method of AKAP4 antigen detection, the detection reagent that the present embodiment is provided with embodiment 3
It is detected, detection method includes the following steps for this；
1.1 take cell concentration be 1 × 106The cell sample of a/mL is washed 1-3 times with PBS sample cleaning solution；
1.2 fix washed cell sample with 3 times of sample fixer again, and 8 DEG C of fixed temperature, time 9min is obtained
Immobilization sample；And immobilization sample is divided into detection fixed sample and control fixed sample；
The antibody for the goat-anti people AKAP4 that 100 μ L are added in 1.3 detection fixed samples obtains mixing sample to be checked, and control is fixed
Sample is added the non-specific IgG of equivalent and obtains control mixing sample；
1.4 mixing samples to be checked and control mixing sample, carry out first respectively under the conditions of 8 DEG C and are incubated for 10min；
The mixing sample to be checked and control mixing sample being incubated in 1.5 steps 1.4 are washed with PBS sample cleaning solution respectively
1-3 times；Then the secondary antibody of the anti-sheep of fluorescein PE label is added；Second is carried out under the conditions of 8 DEG C respectively is incubated for 5min；
1.6 mixing samples to be checked being incubated for through second and control mixing sample are washed 1-3 times with PBS sample cleaning solution, so
After pass through flow cytomery.
Embodiment 6
The present embodiment also provides the detection method of AKAP4 antigen detection, the detection reagent that the present embodiment is provided with embodiment 3
It is detected, detection method includes the following steps for this；
1.1 take cell concentration be 1 × 105The cell sample of a/mL is washed 1-3 times with PBS sample cleaning solution；
1.2 fix washed cell sample with 3 times of sample fixer again, and 6 DEG C of fixed temperature, time 5min is obtained
Immobilization sample；And immobilization sample is divided into detection fixed sample and control fixed sample；
The antibody for the goat-anti people AKAP4 that 50 μ L are added in 1.3 detection fixed samples obtains mixing sample to be checked, compares fixed sample
The non-specific IgG of this addition equivalent obtains control mixing sample；
1.4 mixing samples to be checked and control mixing sample, carry out first respectively under the conditions of 5 DEG C and are incubated for 2h；
The mixing sample to be checked and control mixing sample being incubated in 1.5 steps 1.4 are washed with PBS sample cleaning solution respectively
1-3 times；Then the secondary antibody of the anti-sheep of fluorescein PE label is added；Second is carried out under the conditions of 5 DEG C respectively is incubated for 15min；
1.6 mixing samples to be checked being incubated for through second and control mixing sample are washed 1-3 times with PBS sample cleaning solution, so
After pass through flow cytomery.
Experimental example
The detection method for the AKAP4 antigen detection that this experimental example is provided using embodiment 6 is to the serum sample containing AKAP4
It is detected.Testing result is as shown in Figure 1.
It will be seen from figure 1 that this method can obviously distinguish sample to be examined and check sample, detection effect is obvious, accurately
Reliably.
In conclusion the detection reagent of AKAP4 antigen detection provided by the invention, detection reagent is simple and easy to get, conveniently matches
System；It will test reagent to be applied to prepare the detection kit of AKAP4 antigen detection, facilitate and detected；The detection of AKAP4 antigen
Detection method is easy to operate, and step is simple；It is easily operated；Testing result is accurate and reliable, has preferable practicability.
Embodiments described above is a part of the embodiment of the present invention, instead of all the embodiments.Reality of the invention
The detailed description for applying example is not intended to limit the range of claimed invention, but is merely representative of selected implementation of the invention
Example.Based on the embodiments of the present invention, obtained by those of ordinary skill in the art without making creative efforts
Every other embodiment, shall fall within the protection scope of the present invention.
Claims (10)
- The detection reagent of 1.AKAP4 antigen detection, which is characterized in that the detection reagent mainly includes PBS sample cleaning solution, sample The antibody of this fixer, goat-anti people AKAP4, the secondary antibody of fluorescein-labeled anti-sheep.
- 2. the detection reagent of AKAP4 antigen according to claim 1 detection, which is characterized in that the sample fixer is The PBS buffer solution of paraformaldehyde including 0.1%-0.5%.
- 3. the detection reagent of AKAP4 antigen detection according to claim 1, which is characterized in that the PBS sample cleaning solution PH value with the sample fixer is 6.0-8.0.
- 4. the detection reagent of AKAP4 antigen according to claim 1 detection, which is characterized in that the fluorescein be FITC, One of PE, PI and Percp.
- 5. the detection reagent of AKAP4 antigen detection according to any one of claims 1-4 is in the inspection for preparing the detection of AKAP4 antigen It is applied in test agent box.
- The detection kit of 6.AKAP4 antigen detection, which is characterized in that the detection kit includes PBS sample cleaning solution, sample The antibody of this fixer, goat-anti people AKAP4, the secondary antibody of fluorescein-labeled anti-sheep, sample fixer are the poly for including 0.3% The PBS buffer solution of formaldehyde.
- The detection method of 7.AKAP4 antigen detection, which is characterized in that described detection method includes the following steps:Cell sample is washed with PBS sample cleaning solution, the fixed cell sample of sample fixer of 1-3 times of volume is added, obtains To immobilization sample, the immobilization sample is divided into detection fixed sample and control fixed sample；The antibody of 1-100 μ L goat-anti people AKAP4 is added in the detection fixed sample, obtains mixing sample to be checked；It is described right The non-specific IgG of equivalent is added according to fixed sample, obtains control mixing sample；The mixing sample to be checked and the control mixing sample carry out the first incubation respectively, then respectively with the PBS sample Cleaning solution washing, the secondary antibody that fluorescein-labeled anti-sheep is added carry out second and are incubated for, then washed with the PBS sample cleaning solution It washs；Then pass through flow cytomery.
- 8. the detection method of AKAP4 antigen detection according to claim 7, which is characterized in that the cell sample contains 1 ×104-1×106A/mL cell.
- 9. the detection method of AKAP4 antigen detection according to claim 8, which is characterized in that the fixed cell sample Temperature be 2-8 DEG C, time 4-9min.
- 10. the detection method of AKAP4 antigen detection according to claim 9, which is characterized in that the described first temperature being incubated for Degree is 2-8 DEG C, time 10min-24h；Described second temperature being incubated for is 2-8 DEG C, time 5-30min.
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| CN112630450A (en) * | 2021-01-25 | 2021-04-09 | 安士(广州)医疗科技有限公司 | Preparation method and application of chemiluminescence detection kit for quantitatively detecting concentration of ProAKAP4 |
| CN116655767A (en) * | 2023-07-02 | 2023-08-29 | 广州爱仁生物医药科技有限公司 | Recombinant protein, monoclonal antibody of recombinant protein, and preparation method and application of monoclonal antibody |
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| CN112630450A (en) * | 2021-01-25 | 2021-04-09 | 安士(广州)医疗科技有限公司 | Preparation method and application of chemiluminescence detection kit for quantitatively detecting concentration of ProAKAP4 |
| CN116655767A (en) * | 2023-07-02 | 2023-08-29 | 广州爱仁生物医药科技有限公司 | Recombinant protein, monoclonal antibody of recombinant protein, and preparation method and application of monoclonal antibody |
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Application publication date: 20181225 |