CN109061202A - Dehydroepiandrosterone sulfate chemiluminescence detection kit and preparation method thereof - Google Patents
Dehydroepiandrosterone sulfate chemiluminescence detection kit and preparation method thereof Download PDFInfo
- Publication number
- CN109061202A CN109061202A CN201811113148.0A CN201811113148A CN109061202A CN 109061202 A CN109061202 A CN 109061202A CN 201811113148 A CN201811113148 A CN 201811113148A CN 109061202 A CN109061202 A CN 109061202A
- Authority
- CN
- China
- Prior art keywords
- dehydroepiandrosterone sulfate
- solution
- preparation
- detection kit
- chemiluminescence detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Endocrinology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention provides a kind of dehydroepiandrosterone sulfate chemiluminescence detection kit and preparation method thereof, belongs to immunoassay technical field of medical detection.The kit includes: the dehydroepiandrosterone sulfate antibody of calibration object and quality-control product, Streptavidin magnetic particle suspension liquid, the dehydroepiandrosterone sulfate derivative of acridinium ester label, biotin labeling.The present invention also provides a kind of preparation methods of dehydroepiandrosterone sulfate chemiluminescence detection kit.The kit uses Streptavidin-biotin system, so that signal is further amplified, improves reagent sensitivity.
Description
Technical field
The invention belongs to immunoassay technical field of medical detection, and in particular to a kind of dehydroepiandrosterone sulfate chemiluminescence
Detection kit and preparation method thereof.
Background technique
Dehydroepiandrosterone sulfate (dehydroepiandrosterone sulfate, DHEAs) is dehydrobenzene
The sulfuric acid ester-formin of (dehydroepiandrosterone, DHEA) is one kind mainly by the steroids of adrenocortical secretion
Hormone.As the precursor of male sex hormone and female hormone, DHEAs is the most abundant sterol substance of content in human body, in people's blood
Content ratio DHEA high 250-500 times in clear, it is 100-500 times higher than testosterone, it is 1000-10000 times higher than estradiol.Blood circulation
In DHEAs and do not have male and female hormone biological activity, be transported to after target tissue and generate DHEA through desulfation,
And then it is changed into different male and female hormone compound, play biological function.
The concentration of DHEAs is lower when people is from birth by 5 years old.It was played sexual maturing period from 5 years old, DHEAs gradually rises.To 20-
Reach highest at 30 years old, then gradually decline, the intracorporal content of people is only the 20-30% of peak period when by 70-80 years old.DHEAs
Half-life period in vivo is about one day, and about 1,000 times of the content ratio DHEA high of DHEAs.Due to it high concentration and in a few days and
In the daytime small change rate can be used as the good index of adrenal function.It is more that the raising of detection DHEAs value facilitates diagnosis women
Hair disease and manlike.Other than being diagnosed to hirsutism and Virilism, it may also be used for various masculines, high prolactin blood
Disease, Stein-Leventhal syndrome and the androgen tumour for excluding adrenal cortex generation.
Immunoassay method currently used for detecting dehydroepiandrosterone sulfate mainly has radioimmunology, ELISA
Method, chemiluminescence immunoassay.That there are sensitivity is low for enzyme-linked immunosorbent assay, and the range of linearity is narrow, it is full-automatic to be not easy to realize
The methods of learn limiting factor.Chemiluminescence immunoassay is that the one kind to grow up on the basis of enzyme-linked immunosorbent assay is immunized
Detection technique has high sensitivity, the detection range of linearity wide, easy to operate, the advantages such as the degree of automation height.And strepto- is affine
Element-biotin system has signal amplification, further increases reagent sensitivity.Chemiluminescence immunoassay technology at present
Because it has many advantages, such as above-mentioned be widely used.
Currently, domestic common DHEAs chemiluminescence quantitative detection is mainly international producer Roche, Abbott Laboratories, Siemens, shellfish
The major companies such as Ke Man, internal reagent is less, expensive.Therefore, the DHEAs chemiluminescence detection kit ten of production domesticization is developed
Divide necessity.
Summary of the invention
The object of the present invention is to provide a kind of dehydroepiandrosterone sulfate chemiluminescence detection kits and preparation method thereof, should
Kit sensitivity with higher and accuracy.
Present invention firstly provides a kind of dehydroepiandrosterone sulfate chemiluminescence detection kit, which includes:
Calibration object and quality-control product
Streptavidin magnetic particle suspension liquid
The dehydroepiandrosterone sulfate derivative of acridinium ester label
The dehydroepiandrosterone sulfate antibody of biotin labeling
Preferably, the magnetic bead partial size of the coating Streptavidin is 0.05~3 μm.
Preferably, in the dehydroepiandrosterone sulfate derivative of the acridinium ester label, dehydroepiandrosterone sulfate is derivative
The molar ratio of object and acridinium ester is 1:3-1:20.
Preferably, in the dehydroepiandrosterone sulfate antibody of the biotin labeling, dehydroepiandrosterone sulfate antibody with
The molar ratio of biotin is 1:3-1:15.
Preferably, the concentration of the calibration object is respectively 0 μ g/dL, 1 μ g/dL, 10 μ g/dL, 50 μ g/dL, 250 μ g/
The dehydroepiandrosterone sulfate solution of dL, 500 μ g/dL, 1000 μ g/dL.
Preferably, the concentration of the quality-control product is the quality-control product solution of 5 μ g/dL and 300 μ g/dL.
The present invention also provides a kind of preparation methods of dehydroepiandrosterone sulfate chemiluminescence detection kit, comprising:
Step 1: the preparation of calibration object and quality-control product
Calibration object and quality-control product are prepared with the dehydroepiandrosterone sulfate sterling of human serum matrix；
Step 2: the preparation of Streptavidin magnetic particle suspension liquid
After Streptavidin magnetic particle solution and TBST solution are mixed, be placed on magnetic separator, until supernatant without
Muddiness abandons supernatant, leaves and takes magnetic particle, be made into solid-phase reagent after cleaning in buffer, obtains the suspension of Streptavidin magnetic particle
Liquid；
Step 3: the preparation of the dehydroepiandrosterone sulfate derivative of acridinium ester label
Dehydroepiandrosterone sulfate derivative is put into centrifuge tube, PBS buffer solution centrifugation is then added, it is molten that acridinium ester is added
Liquid stands, then purifies to solution, diluted with buffer, saves；
Step 4: the preparation of the dehydroepiandrosterone sulfate antibody of biotin labeling
Dehydroepiandrosterone sulfate antibody is put into centrifuge tube, PBS buffer solution centrifugation is then added, it is molten that biotin is added
Liquid stands, then purifies to solution, diluted with buffer, saves.
Preferably, the concentration of the PBS buffer solution of the step three and step 4 is 50mM-200mM.
Preferably, the time of repose of the step three and step 4 is preferably 2-4h.
Preferably, the buffer of the step three and step 4 is to contain 1%BSA, and PH is 6.0
100mMbistris buffer.
Beneficial effects of the present invention
The present invention provides a kind of dehydroepiandrosterone sulfate chemiluminescence detection kit and preparation method thereof, the kit packet
It includes: calibration object and quality-control product, Streptavidin magnetic particle suspension liquid, the dehydroepiandrosterone sulfate derivative of acridinium ester label, life
The dehydroepiandrosterone sulfate antibody of object element label；The kit selects acridinium ester for the label material of chemiluminescence immunoassay system
Material, the energy jump which generates when having excitation state to return to ground state are direct chemiluminescence, do not need the participation of enzyme, when saving
Between and cost；The present invention uses Streptavidin-biotin system, compared with existing magnetic bead system, so that signal is further put
Greatly, reagent sensitivity is improved, by changing labelled antibody and antigen, compared with original detection technique, in specific detection human body
Dehydroepiandrosterone sulfate enhances the specificity of detection, provides more accurate, more targetedly in-vitro diagnosis for clinical diagnosis
Reagent.
Detailed description of the invention
Fig. 1 is to survey opposite hair using the detection calibration product examine of dehydroepiandrosterone sulfate chemiluminescence detection kit of the present invention
The standard curve of light value.
Fig. 2 is serum testing result and the Roche company of dehydroepiandrosterone sulfate chemiluminescence detection kit of the present invention
The correlation of kit test result, abscissa X are Roche company kit sample measured value, and concentration is μ g/dL, and ordinate y is
Kit sample value prepared by embodiment 1, concentration unit are μ g/dL.
Specific embodiment
Present invention firstly provides a kind of dehydroepiandrosterone sulfate chemiluminescence detection kit, which includes:
Calibration object and quality-control product
Streptavidin magnetic particle suspension liquid
The dehydroepiandrosterone sulfate derivative of acridinium ester label
The dehydroepiandrosterone sulfate antibody of biotin labeling
According to the present invention, the partial size of Streptavidin magnetic particle is preferably in the Streptavidin magnetic particle suspension liquid
0.05~3 μm, more preferably 3 μm.When the partial size of Streptavidin magnetic particle is lower than 0.05 μm, antigen or antibody and magnetic bead
Percentage bound is low, and it is relatively low to may cause whole light quantity subnumber；It is non-specific when the partial size of Streptavidin magnetic particle is higher than 3 μm
It is obvious in conjunction with effect, it may cause kit poor sensitivity etc..
According to the present invention, in the dehydroepiandrosterone sulfate derivative of the acridinium ester label, dehydroepiandrosterone sulfate spreads out
The molar ratio of biology and acridinium ester is preferably 1:3-1:20, more preferably 1:3.The dehydroepiandrosterone sulfate derivative comes
Source is commercially available, the model DAG1081 selected from the production of Creative Diagnostics company of the U.S..
According to the present invention, in the dehydroepiandrosterone sulfate antibody of the biotin labeling, dehydroepiandrosterone sulfate antibody
Molar ratio with biotin is preferably 1:3-1:15, more preferably 1:3.
According to the present invention, the concentration of the calibration object preferably be respectively 0 μ g/dL, 1 μ g/dL, 10 μ g/dL, 50 μ g/dL,
The dehydroepiandrosterone sulfate solution of 250 μ g/dL, 500 μ g/dL, 1000 μ g/dL；The concentration of the quality-control product is preferably 5 μ g/
The quality-control product solution of dL and 300 μ g/dL.
The present invention also provides a kind of preparation methods of dehydroepiandrosterone sulfate chemiluminescence detection kit, comprising:
Step 1: the preparation of calibration object and quality-control product
DHEAs is diluted to calibration object by employment serum matrix, be distributed into 0 μ g/dL, 1 μ g/dL, 10 μ g/dL, 50 μ g/dL,
250μg/dL,500μg/dL,1000μg/dL；
DHEAs is diluted to the quality-control product that concentration is 5 μ g/dL and 300 μ g/dL by employment serum matrix；
Step 2: the preparation of Streptavidin magnetic particle suspension liquid
After Streptavidin magnetic particle solution and TBST solution are mixed, be placed on magnetic separator, until supernatant without
Muddiness abandons supernatant, leaves and takes magnetic particle, be made into solid-phase reagent after cleaning in buffer, obtains the suspension of Streptavidin magnetic particle
Liquid；
The concentration of the Streptavidin magnetic particle solution is preferably 50~100mg/ml；Streptavidin magnetic particle is molten
The volume ratio of liquid and TBST solution is preferably (0.5~1): (5~10)；The mixing time is preferably 10-15 minutes, described
Buffer be 50mM MES, 0.05% Tween-20,0.05%Proclin300, pH6.5；The concentration of the solid-phase reagent
Preferably 0.05%-0.2%；The source of the Streptavidin magnetic particle solution is commercially available.
Step 3: the preparation of the dehydroepiandrosterone sulfate derivative of acridinium ester label
Dehydroepiandrosterone sulfate derivative is put into centrifuge tube, PBS buffer solution centrifugation is then added, will be delayed in derivative
Fliud flushing is replaced into acridinium ester label buffer, collects solution after displacement, and acridine ester solution is then added, and stands, then to solution
It is purified, is diluted with buffer, saved；
The quality (μ g) of the dehydroepiandrosterone sulfate derivative: the volume (μ l) of PBS buffer solution is preferably 1:1；Institute
The concentration for the PBS buffer solution stated is preferably 50mM-200mM；
The centrifugal rotational speed is preferably 9000-12000rpm, and centrifugation time is preferably 10-20min；Time of repose is preferred
For 2-4h；The molar ratio of the dehydroepiandrosterone sulfate derivative and acridinium ester is preferably 1:3-1:20, more preferably 1:3；
The buffer preferably comprises 1%BSA, and PH is the 100mMbistris buffer of 6.0-8.0；Acridinium ester label after dilution
The concentration of dehydroepiandrosterone sulfate derivative is preferably 0.05-2.0 μ g/ml.
Step 4: the preparation of the dehydroepiandrosterone sulfate antibody of biotin labeling
Dehydroepiandrosterone sulfate antibody is put into centrifuge tube, PBS buffer solution centrifugation is then added, biotin is then added
Solution stands, then purifies to solution, diluted with buffer, saves.
The quality (μ g) of the dehydroepiandrosterone sulfate antibodies Antibodies: the volume (μ l) of PBS buffer solution is preferably 1:1；
The concentration of the PBS buffer solution is preferably 50mM-200mM；
The centrifugal rotational speed is preferably 9000-12000rpm, and centrifugation time is preferably 10-20min；Time of repose is preferred
For 2-4h；The molar ratio of the dehydroepiandrosterone sulfate antibody and biotin is preferably 1:3-1:15, more preferably 1:3；Institute
The buffer stated preferably comprises 1%BSA, and PH is the 100mMbistris buffer of 6.0-8.0；The sulphur of biotin labeling after dilution
The concentration of sour dehydrobenzene antibody is preferably 0.05-2.0 μ g/ml.
According to the present invention, kit detecting step of the invention are as follows: by the sulfuric acid dehydrogenation of sample to be tested and biotin labeling
Epiandrosterone antibody incubation 10min, dehydroepiandrosterone sulfate derivative and the Streptavidin magnetic particle that acridinium ester label is added are outstanding
Supernatant liquid is incubated for 10min, sample and acridinium ester label derivative and biotin labelled antibodies form immune complex, passes through biotin
Magnetic bead is integrated to Streptavidin.By magnetic separator separating immune complexes, cleaning solution washes away unbonded antibody.Pass through acid
Alkali-activated carbonatite liquid excite acridinium ester shine, record relative luminous intensity (RLU), in the linear range, in sample the content of DHEAs with
Relative luminous intensity is inversely proportional.
Further detailed description is done to the present invention combined with specific embodiments below.
Embodiment 1 prepares DHEAs chemiluminescence detection kit of the invention
One, the preparation of calibration object
DHEAs is diluted to calibration object by employment serum matrix, be distributed into 0 μ g/dL, 1 μ g/dL, 10 μ g/dL, 50 μ g/dL,
250μg/dL、500μg/dL、1000μg/dL。
Two, the preparation of quality-control product
DHEAs is diluted to the quality-control product that concentration is 5 μ g/dL and 300 μ g/dL by employment serum matrix.
Three, the preparation of Streptavidin magnetic particle suspension liquid
Taking concentration is 0.5 milliliter of the Streptavidin magnetic particle solution (50mg) of 100mg/ml, and the TBST that 10mL is added is molten
Liquid mixes well after ten minutes, is placed on magnetic separator, until supernatant abandons supernatant, leave and take magnetic particle without muddiness.Repeated washing
Being made into magnetic bead concentration in 50mM MES, 0.05% tween, 0.05%Proclin300, the buffer of pH6.5 after 3 times is
0.2% solid-phase reagent, 4 DEG C of preservations.
Four, the preparation of the dehydroepiandrosterone sulfate R2 solution of acridinium ester label
500 μ g of dehydroepiandrosterone sulfate derivative is added in 10KDa ultra-filtration centrifuge tube, the 100mM PBS of 500ul is added
Buffer, 10000rpm are centrifuged 10min, are repeated 3 times, and are acridinium ester label buffer by buffer exchange in derivative.It collects
Solution after displacement, by derivative: acridinium ester is added in acridinium ester=1:3 molar ratio, stands 2h.Select the full-automatic egg of GE company AKTA
White purifying instrument purifies acridinium ester label derivative solution, collects purified solution, and with 1%BSA is contained, PH is 6.0
Derivative after purification is formulated as the R2 solution of 0.05 μ g/ml, 4 DEG C of preservations by 100mMbistris buffer.
Five, the preparation of the dehydroepiandrosterone sulfate antibody R3 solution of biotin labeling
500 μ g of dehydroepiandrosterone sulfate antibody is added in 50KDa ultra-filtration centrifuge tube, the 100mM PBS that 500ul is added is slow
Fliud flushing, 10000rpm are centrifuged 10min, are repeated 3 times, and are biotin labeling buffer by buffer exchange in antibody.Collect displacement
Solution afterwards, by antibody: biotin is added in biotin=1:3 molar ratio, stands 2h.Select the full-automatic protein purification of GE company AKTA
Instrument purifies biotin labelled antibodies solution, collects purified solution, and with 1%BSA is contained, PH is 6.0
Purified antibodies are formulated as the R3 solution of 0.2 μ g/ml, 4 DEG C of preservations by 100mMBistris buffer.
Six, semi-finished product and finished product composition
The packing of above-mentioned steps products obtained therefrom is semi-finished product.It extracts three parts out and passes through specificity, accuracy, sensitivity, stabilization
After property assay approval, it is assembled into dehydroepiandrosterone sulfate chemical luminescence reagent kit.
Seven, reagent preparation box is examined and determine according to the manufacture and vertification regulation of this field routine, as a result see the table below 1:
Table 1
To sum up, in research process of the invention, the present inventor has carried out screening and quality to raw material used first
It controls, the research of concentration, purity, affinity including antibody, antibody activity after acridinium ester and biotin labeling.Then to acridine
Ester and biotin labeling system are studied, and are tested using not isolabeling ratio and label buffer, by repeatedly real
It tests and comparative experiments, has eventually found that method is easy, high, at low cost, reliable in quality the labeling method of yield.
In addition, the present inventor also explores the reaction pattern of kit and condition.By different anti-
Mode and reaction time comparative experiments are answered, has finally been determined that this kit is incubated for using one step two of competition law, and finally determined
Reagent incubation time is 10min, shortens detection time, improves detection efficiency.
Embodiment 2 prepares DHEAs chemiluminescence detection kit of the invention
One, the preparation of calibration object
DHEAs is diluted to calibration object by employment serum matrix, be distributed into 0 μ g/dL, 1 μ g/dL, 10 μ g/dL, 50 μ g/dL,
250μg/dL、500μg/dL、1000μg/dL。
Two, the preparation of quality-control product
DHEAs is diluted to the quality-control product that concentration is 5 μ g/dL and 300 μ g/dL by employment serum matrix.
Three, the preparation of Streptavidin magnetic particle suspension liquid
Taking concentration is 0.5 milliliter of the Streptavidin magnetic particle solution (50mg) of 100mg/ml, and the TBST that 10mL is added is molten
Liquid mixes well after ten minutes, is placed on magnetic separator, until supernatant abandons supernatant, leave and take magnetic particle without muddiness.Repeated washing
Being made into magnetic bead concentration in 50mM MES, 0.05% tween, 0.05%Proclin300, the buffer of pH6.5 after 3 times is
0.05% solid-phase reagent, 4 DEG C of preservations.
Four, the preparation of the dehydroepiandrosterone sulfate R2 solution of acridinium ester label
500 μ g of dehydroepiandrosterone sulfate derivative is added in 10KDa ultra-filtration centrifuge tube, the 100mM PBS of 500ul is added
Buffer, 10000rpm are centrifuged 10min, are repeated 3 times, and are acridinium ester label buffer by buffer exchange in derivative.It collects
Solution after displacement, by derivative: acridinium ester is added in acridinium ester=1:20 molar ratio, stands 2h.Select GE company AKTA full-automatic
Protein purification instrument purifies acridinium ester label derivative solution, collects purified solution, with containing 1%BSA, PH 8.0
100mM bistris buffer derivative after purification is formulated as to the R2 solution of 0.5 μ g/ml, 4 DEG C of preservations.
Five, the preparation of the dehydroepiandrosterone sulfate antibody R3 solution of biotin labeling
500 μ g of dehydroepiandrosterone sulfate antibody is added in 50KDa ultra-filtration centrifuge tube, the 100mM PBS that 500ul is added is slow
Fliud flushing, 10000rpm are centrifuged 10min, are repeated 3 times, and are biotin labeling buffer by buffer exchange in antibody.Collect displacement
Solution afterwards, by antibody: biotin is added in biotin=1:15 molar ratio, stands 2h.Select the full-automatic albumen of GE company AKTA pure
Change instrument to purify biotin labelled antibodies solution, collects purified solution, with containing 1%BSA, the 100mM that PH is 8.0
Purified antibodies are formulated as the R3 solution of 2 μ g/ml, 4 DEG C of preservations by Bistris buffer.
The methodology of the DHEAs chemiluminescence detection kit of the invention of embodiment 3 is examined and determine
The kit prepared in embodiment 1 is examined and determine according to professional standard in this field and vertification regulation, as a result such as
Under:
1. the kit range of linearity measures
By the kit prepared in embodiment 1 wherein 1 batch, measurement 0 μ g/dL of range of linearity sample, 1 μ g/dL, 10 μ g/dL,
50 μ g/dL, 250 μ g/dL, 500 μ g/dL, 1000 μ g/dL show that the range of linearity is 0-1000 μ g/dL, coefficient R 2=
0.9999, standard curve is as shown in Figure 1, the results are shown in Table 2:
Table 2
2. the measurement of kit sensitivity
By the kit prepared in embodiment 1 wherein 1 batch, 20 neck concentration samples are measured, calculate average value (M) and standard
Poor (SD), obtains M-2SD, carries out two o'clock regression fit according to the concentration-RLU between zero-dose calibration object and adjacent calibration object and obtains
Linear function out brings the RLU of M-2SD into equation, show that respective concentration value is 0.59 μ g/dL of kit sensitivity.
1st luminous value is as shown in table 3:
Table 3
First point of shine mean value M=523765, SD=3960.6, M-2SD=515844
2nd luminous value is as shown in table 4:
Table 4
First point and the matched curve of second point line, sensitivity=0.59 μ g/dL of calculating.
3. kit specificity experiments, as shown in table 5:
Table 5
By a large amount of repeated experiments, kit index of the invention is as follows:
Detection range: 0.6-1000 μ g/dL；
Sensitivity: minimum detection limit is not higher than 0.6 μ g/dL；
Specificity: it is 0.08% with dehydrobenzene (DHEA) cross reacting rate, is with androstenedione cross reacting rate
0.05%, it is 0.02% with testosterone (T) cross reacting rate, is 0.01% with estradiol (E2) cross reacting rate, intersects with oestrone
Reactivity is 0.01%
The kit of the invention of embodiment 4 is compared with external kit clinical sample measured value
Clinical serum is detected simultaneously with the kit and Roche company kit prepared in embodiment 1.Its testing result is shown in
Fig. 2, using the result that Roche kit measures as abscissa X, concentration is μ g/dL, is sat using the result that the present invention measures as vertical
Y is marked, concentration unit is μ g/dL, makees regression equation, dependent equation are as follows: y=0.999x-0.297, related coefficient are 0.985. warp
Statistical procedures the result shows that, this method and external kit clinical correlation are good.
Claims (10)
1. a kind of dehydroepiandrosterone sulfate chemiluminescence detection kit, which is characterized in that the kit includes:
Calibration object and quality-control product
Streptavidin magnetic particle suspension liquid
The dehydroepiandrosterone sulfate derivative of acridinium ester label
The dehydroepiandrosterone sulfate antibody of biotin labeling.
2. a kind of dehydroepiandrosterone sulfate chemiluminescence detection kit according to claim 1, which is characterized in that described
Coating Streptavidin magnetic bead partial size be 0.05~3 μm.
3. a kind of dehydroepiandrosterone sulfate chemiluminescence detection kit according to claim 1, which is characterized in that described
Acridinium ester label dehydroepiandrosterone sulfate derivative in, the molar ratio of dehydroepiandrosterone sulfate derivative and acridinium ester is 1:
3-1:20。
4. a kind of dehydroepiandrosterone sulfate chemiluminescence detection kit according to claim 1, which is characterized in that described
Biotin labeling dehydroepiandrosterone sulfate antibody in, the molar ratio of dehydroepiandrosterone sulfate antibody and biotin is 1:3-1:
15。
5. a kind of dehydroepiandrosterone sulfate chemiluminescence detection kit according to claim 1, which is characterized in that described
The concentration of calibration object be respectively 0 μ g/dL, 1 μ g/dL, 10 μ g/dL, 50 μ g/dL, 250 μ g/dL, 500 μ g/dL, 1000 μ g/dL
Dehydroepiandrosterone sulfate solution.
6. a kind of dehydroepiandrosterone sulfate chemiluminescence detection kit according to claim 1, which is characterized in that described
Quality-control product concentration be 5 μ g/dL and 300 μ g/dL quality-control product solution.
7. a kind of preparation method of dehydroepiandrosterone sulfate chemiluminescence detection kit according to claim 1, special
Sign is, comprising:
Step 1: the preparation of calibration object and quality-control product
Calibration object and quality-control product are prepared with the dehydroepiandrosterone sulfate sterling of human serum matrix；
Step 2: the preparation of Streptavidin magnetic particle suspension liquid
After Streptavidin magnetic particle solution and TBST solution are mixed, it is placed on magnetic separator, until supernatant is without muddiness,
Supernatant is abandoned, magnetic particle is left and taken, is made into solid-phase reagent after cleaning in buffer, obtain Streptavidin magnetic particle suspension liquid；
Step 3: the preparation of the dehydroepiandrosterone sulfate derivative of acridinium ester label
Dehydroepiandrosterone sulfate derivative is put into centrifuge tube, PBS buffer solution centrifugation is then added, acridine ester solution is added,
It stands, then solution is purified, is diluted with buffer, save；
Step 4: the preparation of the dehydroepiandrosterone sulfate antibody of biotin labeling
Dehydroepiandrosterone sulfate antibody is put into centrifuge tube, PBS buffer solution centrifugation is then added, biotin solution is added, it is quiet
It sets, then solution is purified, diluted with buffer, saved.
8. a kind of preparation method of dehydroepiandrosterone sulfate chemiluminescence detection kit according to claim 7, special
Sign is that the concentration of the PBS buffer solution of the step three and step 4 is 50mM-200mM.
9. a kind of preparation method of dehydroepiandrosterone sulfate chemiluminescence detection kit according to claim 7, special
Sign is that the time of repose of the step three and step 4 is preferably 2-4h.
10. a kind of preparation method of dehydroepiandrosterone sulfate chemiluminescence detection kit according to claim 7, special
Sign is that the buffer of the step three and step 4 is to contain 1%BSA, the 100mMbistris buffer that PH is 6.0.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811113148.0A CN109061202A (en) | 2018-09-25 | 2018-09-25 | Dehydroepiandrosterone sulfate chemiluminescence detection kit and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811113148.0A CN109061202A (en) | 2018-09-25 | 2018-09-25 | Dehydroepiandrosterone sulfate chemiluminescence detection kit and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109061202A true CN109061202A (en) | 2018-12-21 |
Family
ID=64763525
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811113148.0A Pending CN109061202A (en) | 2018-09-25 | 2018-09-25 | Dehydroepiandrosterone sulfate chemiluminescence detection kit and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109061202A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109991203A (en) * | 2019-04-18 | 2019-07-09 | 上海大学 | It is a kind of for detecting the kit and method of Ochratoxin A |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102565405A (en) * | 2011-08-24 | 2012-07-11 | 苏州长光华医生物试剂有限公司 | Method for immunological detection by combining acridinium ester labeling technology with general magnetic particles |
| CN105190311A (en) * | 2013-03-15 | 2015-12-23 | 雅培实验室 | Assay with increased dynamic range |
-
2018
- 2018-09-25 CN CN201811113148.0A patent/CN109061202A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102565405A (en) * | 2011-08-24 | 2012-07-11 | 苏州长光华医生物试剂有限公司 | Method for immunological detection by combining acridinium ester labeling technology with general magnetic particles |
| CN105190311A (en) * | 2013-03-15 | 2015-12-23 | 雅培实验室 | Assay with increased dynamic range |
Non-Patent Citations (2)
| Title |
|---|
| B. KUPELI ET AL: "The role of cigarette smoking in prostatic enlargement", 《BRITISH JOURNAL OF UROLOGY》 * |
| 苏立等: "硫酸去氢表雄酮定量测定试剂盒(化学发光免疫分析法)的研制及性能评价", 《分子诊断与治疗杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109991203A (en) * | 2019-04-18 | 2019-07-09 | 上海大学 | It is a kind of for detecting the kit and method of Ochratoxin A |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA1040082B (en) | Process for the demonstration and determination of reaction components having specific binding affinity for each other | |
| USRE29169E (en) | Process for the demonstration and determination of reaction components having specific binding affinity for each other | |
| USRE31006E (en) | Process for the demonstration and determination of reaction components having specific binding affinity for each other | |
| CN100464189C (en) | Magnetic separating direct chemical illuminating reagent and testing method using the same reagent | |
| CN103472229B (en) | A kind of carcinomebryonic antigen magnetic microparticle chemiluminescence immune assay detection kit | |
| CN107543932A (en) | The magnetic microparticle chemiluminescence detection kit and preparation method of a kind of calcitonin | |
| CN109001472A (en) | Human thyrotropin receptor antibody chemical luminescence detection kit and preparation method thereof and application method | |
| CN107817232A (en) | For the automation immunoassay system for the diagnostic assay for carrying out allergy and autoimmune disease | |
| CN109633180A (en) | A kind of free testosterone chemiluminescence detection kit and preparation method thereof | |
| CN102159591A (en) | Monoclonal antibody, and immunoassay using same | |
| CN103033619A (en) | Protein chip reagent kit and method for comprehensively detecting lung cancer marker | |
| CN106918708A (en) | A kind of competition law turbid kit of latex enhancing immune transmittance for detecting insulin | |
| CN108169486A (en) | A kind of kit and its test method for measuring squamous cell carcinoma-related antigen content | |
| CN111175494A (en) | Thyroglobulin antibody detection kit and use method thereof | |
| CN107271685A (en) | A kind of method and its dedicated kit for detecting the content of fowl leukocyte interleukin 2 | |
| JP2023017986A (en) | Direct immunoassay measurement of autoantibodies | |
| CN109001471A (en) | Free beta-human chorionic gonadotropin chemiluminescence detection kit and preparation method thereof and application method | |
| CN107860914A (en) | A kind of anti-mitochondrial antibody IgG detection kit and its detection method | |
| CN111579781A (en) | Hepatitis C virus antibody detection kit, preparation method and detection method | |
| CN110672850A (en) | Hepatitis A virus antibody IgM detection kit and preparation method thereof | |
| CN109061202A (en) | Dehydroepiandrosterone sulfate chemiluminescence detection kit and preparation method thereof | |
| CN110988325B (en) | Blocking agent and kit containing same | |
| CN109239359A (en) | β 2-MG chemical luminescence immune assay determination reagent kit and preparation method thereof | |
| CN109085343B (en) | Kit for determining anti-Jo-1 antibody and detection method | |
| CN101545910A (en) | Ferritin chemoluminescence immunoassay quantitative measuring kit and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20181221 |
|
| WD01 | Invention patent application deemed withdrawn after publication |