CN106501535A - A kind of compound releasing agent of ANS salt and the kit for detecting Blood cortisol - Google Patents
A kind of compound releasing agent of ANS salt and the kit for detecting Blood cortisol Download PDFInfo
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- CN106501535A CN106501535A CN201611082658.7A CN201611082658A CN106501535A CN 106501535 A CN106501535 A CN 106501535A CN 201611082658 A CN201611082658 A CN 201611082658A CN 106501535 A CN106501535 A CN 106501535A
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- releasing agent
- acridinium ester
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- 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
- G01N33/743—Steroid hormones
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- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
Abstract
The present invention discloses the kit of a kind of compound releasing agent of ANS salt and detection Blood cortisol, and kit includes following components：The compound releasing agent of the magnetic bead of coated cortisol antibody, the cortisol BSA compounds of acridinium ester label, ANS salt and cortisol calibration object.The cortisol of combining form in blood of human body sample is changed into ANS salt of the present invention compound releasing agent the cortisol of free form, and detects the content of free cortisol by acridinium ester chemiluminescent.The compound releasing agent of ANS salt can be combined from different glucocorticosteroid albumen, can fully be discharged the cortisol in blood with reference to state.Acridinium ester chemiluminescent is luminous for flash type, few during illuminating, photon that can completely produced by catching reaction；Acridinium ester is artificial synthetic, and the anti-interference to sample interfering material is higher, detects that using acridinium ester label the content of free cortisol is very sensitive, makes the assay of total cortisol in blood sample more accurate.
Description
Technical field
The present invention relates to technical field of immunoassay, more particularly to a kind of compound releasing agent of ANS salt and skin in detection blood
The kit of matter alcohol.
Background technology
Cortisol (Cortisol) also known as " hydrocortisone ", is a kind of sugared cortex that adrenal gland is produced in stress reaction
Hormone.When people takes in cholesterol from food, in the presence of body produces stress reaction, during cholesterol is via adrenal gland
" glucocorticoid system " becomes cortisol, and is secreted in blood in a free form.Cortisol has regulation carbon hydrate
The function of thing metabolism；Immunosupress and the effect of anti-inflammatory；To hypothalamus secrete corticoliberim and hang down
The corticotropin of body secretion plays negative-feedback regu- lation effect.Therefore, by the content of detection cortisol, kidney can be diagnosed
The functional status of upper gland, while can be with the functional status of indirect observation hypophysis.
Can be with free cortex in blood due to there is substantial amounts of corticosteroid globulin and albumin in blood of human body
Alcohol is combined, and the cortisol in conjunction with after can not be from glomerular filtration, and now cortisol just loses biologically active.And it is only a small amount of
Cortisol is present in a free form, and free cortisol can be leached through glomerulus, enters in urine.Therefore want accurately
The content of cortisol in blood sample is determined, free cortisol how is discharged and is just particularly important.
The releasing agent that selects on the market at present is mainly DANAZOL, and its structural formula is as follows：
DANAZOL (Danazol) is a kind of artificial synthesized steroidal heterocyclic compound, i.e. androgenous 11 7a- Ethisterones
Derivative, be white or creamy white crystals or crystalline powder.Due to water insoluble, it is necessary to by organic solvent, such as DMF
(dimethylformamide), chloroform, acetone etc. dissolve.But these organic solvents can affect cortisol to discharge, so as to cause blood
In dissociate Determination of cortisol detection inaccurate.Therefore select not only to be dissolved in water but also play the releasing agent of cortisol release action
It is particularly important.
The method of current cortisol immunodiagnosis mainly has following several method：
1. the Direct Electrochemistry of different luminol and its derivative substance markers lights
Principle：With different luminol and its derivative as luminous substrate, reaction speed is improved using the catalyst of metal ion
Degree and sensitivity, use NaOH+H2O2Make luminous exciting liquid, the direct chemiluminescence of flash type is whole using photon counter collection
Course of reaction lights the measuring method of counting.
Not enough：Different luminol and its derivative light as flash type, and its luminous intensity depended in immune response
The concentration of enzyme, if not using, catalyst or catalytic amount are not enough, luminous signal dies down, and the amount of catalyst is difficult to control；Its
Secondary, different luminol by sample in haemolysis disturb, affect blood in dissociate Determination of cortisol measure.
2. electrochemical luminescence
Principle：Tris (bipyridine) ruthenium [Ru (bpy) of the ECLI method luminescence reagents label for divalence3]2+, another reagent is 3 third
Amine TPA, can lose electronics simultaneously in anode surface both the above electroactive substance and oxidized.The tripropyl amine (TPA) of oxidation loses
One H+And become strong reductant, by the tris (bipyridine) ruthenium [Ru (bpy) of oxidized form3]3+It is reduced to [the Ru (bpy) of excitation state3
]2+, discharging photon immediately and recover the luminous substrate of ground state, this process is carried out again and again in electrode surface, is constantly sent
Photon and often keep the constant of concentration of substrate.
Not enough：Excessively sensitive to environmental factor and other nonspecific reactions；For metal ion, EDTA or other anti-freezings
Agent, preservative metallo-chelate excessively sensitive；The deposition of reaction by-product is also a potential interference source；To beyond pH being
The sample of 6-7 is very sensitive；O2In the case where voltage conditions are added, light under the conditions of 620nm, electrochemical luminescence is by reaction system
The interference of dissolved oxygen；Wiper mechanism flow cell is cleaned, and flow cell recycles, for cleaning requirement higher.
3. luminol-HRP systems
Principle：With luminol as luminous substrate in the basic conditions, formed a pairs of anion.This pairs of anion can quilt
Dioxygen oxidation produces a highly unstable organic peroxide.The peroxide is very unstable, decomposites nitrogen immediately, raw
3- aminophthalic acids into triplet state (excitation state).When the latter recovers to ground state, launch photon, about 20 points of continuous illumination
Clock is longer.
Not enough：HRP is by heavy metal ion, temperature, NaN in sample3Disturb etc. factor；Reagent stability is subject to sample pilot scale
Agent stability is restricted by HRP stability；Label is protein molecular, and label is added and increased antigen-antibody reaction steric hindrance.
4. adamantane-ALP systems
Principle：Unboiled water solution is issued as luminous substrate in alkaline phosphatase enzyme effect with spiral adamantane, obtain one medium
Stable intermediate A MPD-(2～30 minutes half-life).In this intermediate molecule, electro transfer is cracked into the adamantane of a molecule
Ketone and molecule oxybenzoic acid methyl esters anion between excitation state, produces the light of 470nm, and continues when which returns to ground state
Dozens of minutes, belongs to wide variety of glow-type enzyme-catalyzed chemical luminescence, and intercepting one section fluorescent lifetime using photon counter carries out count measurement.
Not enough：ALP is disturbed by the factor such as heavy metal ion, temperature in sample；Reagent stability is subject to reagent in sample steady
Qualitative restricted by ALP stability；Label is protein molecular, and label is added and increased antigen-antibody reaction steric hindrance.
From the foregoing, there is drawback in its clinical practice of existing external diagnosis reagent at present.
Therefore, prior art has yet to be improved and developed.
Content of the invention
In view of above-mentioned the deficiencies in the prior art, it is an object of the invention to provide a kind of compound releasing agent of ANS salt and detection
The kit of Blood cortisol, it is intended to solve the problems referred to above that existing external diagnosis reagent is present.
Technical scheme is as follows：
A kind of kit of detection Blood cortisol, wherein, including following components：The magnetic bead of coated cortisol antibody,
The compound releasing agent of the cortisol BSA compounds of acridinium ester label, ANS salt and cortisol calibration object.
The kit of described detection Blood cortisol, wherein, also includes luminous cleaning fluid A and luminous cleaning fluid B.
The kit of described detection Blood cortisol, wherein, the magnetic bead be coated with carboxyl, epoxy radicals,
Any one in toluene Huang acyl group.
The kit of described detection Blood cortisol, wherein, the compound substance markers of acridinium ester and cortisol BSA mole
Ratio is between 1:2.5-1:Between 10.
The kit of described detection Blood cortisol, wherein, the cortisol BSA compounds of the acridinium ester label are
Cortisol-3-BSA compounds.
The kit of described detection Blood cortisol, wherein, the cortisol calibration object is the alcohol hemocrinia that decorticates
The thin cortisol antigen that releases.
The kit of described detection Blood cortisol, wherein, the luminous cleaning fluid A includes following components：50-
The disodium hydrogen phosphate of 100g/L, the sodium dihydrogen phosphate of 5-30g/L, the sodium chloride of 50-200g/L, the Tween- of 0.1%-10%
20th, the hydrogen peroxide and the nitric acid of 1-2M of liquid bio preservative Proclin-300,0.5%-5% of 0.1%-10%.
The kit of described detection Blood cortisol, wherein, the luminous cleaning fluid B includes following components：50-
The disodium hydrogen phosphate of 100g/L, the sodium dihydrogen phosphate of 5-30g/L, the sodium chloride of 50-200g/L, the Tween- of 0.1%-10%
20th, the NaOH and 0.1%-2% of liquid bio preservative Proclin-300,0.01-2.5M of 0.1%-10%
TritonX-100.
A kind of ANS salt is combined releasing agent, and wherein, the compound releasing agent of the ANS salt includes following components：3-15mg/ml
ANS salt, 0.1-0.5%NaN3, 100-200mM NaCl and 20-60mM barbital sodiums.
The compound releasing agent of described ANS salt, wherein, the ANS salt has following structure：
Above-mentioned R+For NH4 +、K+Or Na+.
Beneficial effect：The cortisol of combining form in blood of human body sample is turned by the present invention by the compound releasing agent of ANS salt
Be changed into the cortisol of free form, and the content of free cortisol is detected by chemiluminescent new technique.From ANS salt
Compound releasing agent can be combined from different glucocorticosteroid albumen, can fully be discharged the cortisol in blood with reference to state
Come；Described chemiluminescence is acridinium ester chemiluminescent, and acridinium ester chemiluminescent lights for flash type, few during illuminating, can
Photon completely produced by catching reaction；Acridinium ester is artificial synthetic, and the anti-interference to sample interfering material is higher, profit
Detect that with acridinium ester label the content of free cortisol is very sensitive, make the assay of total cortisol in blood sample more be defined
Really.
Description of the drawings
Theoretical concentration and actually detected concentration relationship schematic diagram of the Fig. 1 for the embodiment of the present invention.
Specific embodiment
The present invention provides the kit of a kind of compound releasing agent of ANS salt and detection Blood cortisol, for making the present invention's
Purpose, technical scheme and effect are clearer, clear and definite, and the present invention is described in more detail below.It should be appreciated that this place is retouched
The specific embodiment that states only in order to explain the present invention, is not intended to limit the present invention.
The present invention provides a kind of ANS salt compound releasing agent, and wherein, the compound releasing agent of the ANS salt includes following components：3-
15mg/ml ANS salt, 0.1-0.5%NaN3, 100-200mM NaCl and 20-60mM barbital sodiums.
ANS salt (8-anilino-1-naphthalene sulfonic acid salt) is a kind of fluorescent dye, the hydrophobic surface of protein is had high affine
Power.ANS salt can be attached to the protein surface of low polar region.It is larger that these attributes make ANS salt be highly suitable for affinity
Hydrophobic ligand and its corresponding associated proteins.And cortisol be small molecule parahormone, generally in blood with associated proteins
In conjunction with, and cortisol is combined with biologically active, free cortisol can be made to obtain sufficiently using ANS salt releasing agent is added
Release；Compared to traditional releasing agent DANAZOL, ANS salt need not dissolve in organic solvent, directly can dissolve in water,
Operating process is reduced, is reduced reagent raw material consumables cost, and ANS salt is much larger than to the actual release efficiency of free cortisol and reaches
That azoles effectively improves sensitivity and the degree of accuracy to its release efficiency.
Wherein, the structural formula of the barbital sodium is as follows,
Barbital sodium (BBTS) is a kind of chemical substance, and, with electrostatic force interaction, the process is for barbital sodium and albumen
The spontaneous supermolecular mechanism process that one entropy increase, BBTS free energys reduce.For the life such as the small molecules such as cortisol and protein
Active force type between thing macromolecular has hydrophobic force, hydrogen bond, Van der Waals force and electrostatic force.Under uniform temperature and pressure, egg
The space subdomain of white macromolecular forms cylinder-like structure in the way of notch is relative, and almost all of hydrophobic amino acid is residual
Base is all embedded in cylinder interior, constitutes hydrophobic pocket, and there is a kind of electrostatic force between BBTS and albumen so that BBTS molecules
With the binding site of albumen in this columnar hydrophobic pocket, the cortisol small molecule that combine can part is released.
And ANS salt actions are in the larger hydrophobic ligand of affinity and its corresponding associated proteins hydrophobic surface.Release when being combined in ANS salt
Put in agent and add ANS salt and BBTS, free cortisol in associated proteins can be made more fully to be discharged.
The present invention also provides a kind of kit preferred embodiment of detection Blood cortisol, wherein, including following components：
The compound releasing agent of the magnetic bead of coated cortisol antibody, the cortisol BSA compounds of acridinium ester label, ANS salt as above with
Cortisol calibration object.Preferably, the kit of present invention detection Blood cortisol also includes luminous cleaning fluid A and luminous cleaning
Liquid B.
Specifically, the magnetic bead of the coated cortisol antibody of the present invention, wherein, the particle size of the magnetic bead is 1.5 μm of -2.8 μ
m.Preferably, the kernel of the magnetic bead be ferroso-ferric oxide, the magnetic bead be coated with carboxyl, epoxy radicals or toluene Huang acyl
Any one in base.Magnetic bead can affect accuracy using the incomplete sedimentation of front solid phase, should select good dispersion when therefore selecting,
It is few that long-time places magnetic bead number of uniting, the slow magnetic bead of sinking speed.And the magnetic bead with ferroso-ferric oxide as kernel, its surface bag
The variety classes group hydrophily that wraps up in is preferable so that can effectively carry out separating between magnetic bead, wash, settling flux, can drop significantly
Low non-specific adsorption, can greatly improve precision when reaction is really achieved equilibrium condition.Preferably, coated cortisol antibody
Magnetic bead in, according to 1～2% ratio coating cortisol antibody.
Specifically, the cortisol BSA compounds of acridinium ester label of the present invention, wherein, acridinium ester is multiple with cortisol BSA
The molar ratio of compound mark is between 1:2.5-1:Between 10, preferred molar ratio example is 1:5.Select good hydrophilic property (non-specific
Absorption is little) acridinium ester, free non-specific adsorption between acridinium ester and solid-phase magnetic beads can be reduced, can be with by chemical modification
Change the hydrophobicity of acridinium ester, reduce the purpose of non-specific adsorption.Preferably, the skin of acridinium ester label of the present invention
Matter alcohol BSA compounds are Cortisol-3-BSA compounds.
Specifically, the cortisol calibration object be decorticate alcohol hormone serum-dilution cortisol antigen concentration be 0.08 μ
The solution of g/L-6 μ g/L.
Specifically, the luminous cleaning fluid A includes following components：The disodium hydrogen phosphate of 50-100g/L, the phosphorus of 5-30g/L
Acid dihydride sodium, the sodium chloride of 50-200g/L, the liquid bio preservative of Tween-20,0.1%-10% of 0.1%-10%
The nitric acid of the hydrogen peroxide and 1-2M of Proclin-300,0.5%-5%, percentage composition is weight/mass percentage composition.For example, above-mentioned
Luminous cleaning fluid A can include following components：The disodium hydrogen phosphate of 70g/L, the sodium dihydrogen phosphate of 10g/L, the chlorination of 180g/L
Sodium, 10% Tween-20, the nitric acid of 2% liquid bio preservative Proclin-300,4% hydrogen peroxide and 1M, percentage
Content is weight/mass percentage composition.
Specifically, the luminous cleaning fluid B includes following components：The disodium hydrogen phosphate of 50-100g/L, the phosphorus of 5-30g/L
Acid dihydride sodium, the sodium chloride of 50-200g/L, the liquid bio preservative of Tween-20,0.1%-10% of 0.1%-10%
The NaOH and 0.1%-2%TritonX-100 of Proclin-300,0.01-2.5M, percentage composition is weight/mass percentage composition.
For example, above-mentioned luminous cleaning fluid B can include following components：The disodium hydrogen phosphate of 70g/L, the sodium dihydrogen phosphate of 10g/L,
The sodium chloride of 180g/L, 10% Tween-20, the NaOH of 2% liquid bio preservative Proclin-300,1.5M and
1%TritonX-100, percentage composition are weight/mass percentage composition.
Below by embodiment, the present invention is described in detail.
Embodiment
1st, prepared by the Myoglobin reagent of coated cortisol antibody
1), by 5mg particle diameters be 2.0 μm of magnetic beads, with 50mM pH for 6.0 ± 0.1 MES buffer solutions resuspended, shaken using whirlpool
Device mixing is swung, by centrifuge tube (note after fully shaking：Centrifugation lid should not have magnetic bead to remain) it is placed on Magneto separate frame, Zhi Daoshang
Clear liquid is completely thorough, is carefully moved with micropipettor and abandons supernatant, is repeated twice.
2), with 50mM pH for 6.0 ± 0.1 the resuspended magnetic bead of MES buffer solutions to target volume, vortex oscillator is mixed, is filled
Appropriate cortisol antibody is added according to 1.5% coating ratio after dividing concussion, is fully mixed using vortex oscillator rapidly, Ran Houjia
Enter MES buffer solutions and 5M ammonium sulphate buffers that 50mM pH are 6.0 ± 0.1, fully mixed using vortex oscillator rapidly,
Coating is shaken up overnight using vortex mixer under the conditions of 37 DEG C.
3), after, reaction terminates, centrifuge tube (centrifugation lid there should not be magnetic bead to remain) is placed on Magneto separate frame, until upper
After clear liquid is thorough, is carefully moved with micropipettor and abandon supernatant, add 1mL confining liquids, the confining liquid is containing 5% (W/V) BSA
50mM pH for 6.0 ± 0.1 MES buffer solutions, 37 DEG C of conditions using vortex mixer shake up closing 14h.
4), centrifuge tube (centrifugation lid should not have magnetic bead remain) is placed on Magneto separate frame, until supernatant completely saturating
After thorough, carefully moved with micropipettor and abandon supernatant, the use of the 10mmol pH containing 8% (w/v) BSA is 7.4 ± 0.1 at 37 DEG C
PB buffer solution for cleaning liquid cleans magnetic bead, and centrifuge tube (centrifugation lid should not have magnetic bead to remain) is placed on by magnetic bead concentration 1mg/mL
On Magneto separate frame, after supernatant becomes completely thorough, carefully moved with micropipettor and abandon supernatant, repeated washing 3 times, every time 1
Hour.
5), with the resuspended magnetic bead of the poloxamer reagent buffer for containing 0.2%, vortex oscillator is fully mixed, 2-8 DEG C of guarantor
Deposit.
2nd, prepared by the cortisol BSA compounds liquid-phase reagent of acridinium ester label
1), deionized water prepares the 20mM PBS acridinium ester purification buffers containing 2%BSA.
2) the cortisol BSA compounds of 50 μ g, are added, and adds 100 μ L acridinium ester purification buffers.
3), the molar ratio for being combined substance markers according to acridinium ester and cortisol BSA is 1:5 ratio, adds 0.25-1 μ L
Acridinium ester (2mg/mL).
4), it is put in light culture case, 37 DEG C of shaking tables are mixed, light culture 2h.
5), blocking agent 1h is added.
6), the cortisol BSA complex purifications that mark is completed.
7), by mass percentage, 0.25-1 μ g/mL are diluted to 0.2% poloxamer reagent buffer marking fluid
Working solution.
3rd, prepared by the compound releasing agent of ANS salt
By mass percentage, with 5mg/ml ANS-Na (8-anilino-1-naphthalene sulfonic acid sodium salt), 0.1%NaN3、150mM
The compound releasing agent of ANS salt prepared by NaCl and 40mM barbital sodiums, adjusts pH to 7.0, is packed as one bottle per 100mL, is placed in 4 DEG C of guarantors
Deposit.
4th, prepared by cortisol calibration object
1), the alcohol hormone serum that decorticates of 1000mL is placed in 37 DEG C of thermostat water baths, is inactivated.
2), by mass percentage, with 150mM NaCl, 1%Proclin-300 and 0.1%NaN3After adding fire extinguishing
Decorticate in alcohol hormone serum, one bottle is packed as per 100mL, is placed in 4 DEG C of preservations.
3), by cortisol antigen sterling with the above-mentioned alcohol hormone serum buffer doubling dilution that decorticates for preparing:0.093、
0.214th, 0.493,1.134,2.609,6 μ g/L totally 6 bottles of calibration objects.
5th, light cleaning fluid A, B preparation
1), light cleaning fluid A：The disodium hydrogen phosphate of addition 70g/L, the sodium dihydrogen phosphate of 10g/L, the chlorination of 180g/L
Sodium, 10% Tween-20, the nitric acid of 2% liquid bio preservative Proclin-300,4% hydrogen peroxide and 1M, will be upper
State material to be sufficiently mixed, obtain luminous cleaning fluid A.
2), light cleaning fluid B：The disodium hydrogen phosphate of addition 70g/L, the sodium dihydrogen phosphate of 10g/L, the chlorination of 180g/L
Sodium, 10% Tween-20, the NaOH of 2% liquid bio preservative Proclin-300,1.5M and 1%TritonX-
100, above-mentioned substance is sufficiently mixed, luminous cleaning fluid B is obtained.
Myoglobin reagent, acridine of the free cortisol kit by above-mentioned coated cortisol antibody in efficient detection blood
The cortisol BSA compound liquid-phase reagents of ester mark, ANS salt are combined releasing agent, cortisol calibration object, the cleaning fluid A that lights, light
Cleaning fluid B is constituted.
Project standard see the table below 1.
Table 1, project standard
Testing result is as follows：
1st, the range of linearity：
Logit-log models fittings are used, concentration range is 0-6 μ g/L, for sample of the concentration more than 6 μ g/L first should be carried out
It is measured after dilution again；Linear coefficient：R2≥0.99；As shown in table 2 below, accompanying drawing 1.
Table 2, Concentration Testing result statistical form
2. sensitivity for analysis:
Detected with the matrix without measured object, replication 20 times, shown that the RL Μ values of 20 testing results are (relative
Luminous value), its mean value (M) and standard deviation (SD) is calculated, the RL Μ values corresponding to M-2SD are drawn, according to without measured object
Concentration-RL Μ values results between matrix and low value calibration object carry out 2 points of regression fits and draw linear function, will be right for M-2SD institutes
The RL Μ values that answers are brought in above-mentioned equation, obtain corresponding concentration value, as sensitivity for analysis, and its result should meet≤0.08 μ
G/L, data are as shown in table 3 below.
Table 3, sensitivity for analysis testing result statistical form
3rd, precision：
1) precision in, analyzing：2 supporting quality-control products of test kit, i.e. low value quality-control product and high level quality-control product,
In same analysis, each quality-control product duplicate detection 10 times calculates mean value (M) and the standard deviation (SD) of 10 measurement results, presses
Equation below (1) calculates the coefficient of variation (CV), and the test result coefficient of variation (CV) answers≤8.0%, and data are as shown in table 4 below.
2) precision between, analyzing：With 3 batch kits, 2 quality-control products that is tested in same set of kit respectively, i.e.,
Low value quality-control product and high level quality-control product, each each sample of batch kit duplicate detection 10 times calculate the flat of 10 measurement results
Average (M) and standard deviation (SD), as follows (1) calculate the coefficient of variation (CV), then batch anaplasia between 3 lot number kits
Different coefficient (CV)≤15.0%, data are as shown in table 5 below.
Formula is as follows：
CV=SD/M × 100% ... ... ... ... (1)
In formula：The CV coefficient of variation；
The standard deviation of SD measurement results；
The mean value of M measurement results.
Precision testing result statistical form in table 4, analysis
| Precision in analysis | Quality-control product low value (μ g/L) | Quality-control product high level (μ g/L) |
| 1 | 0.310 | 2.903 |
| 2 | 0.309 | 2.976 |
| 3 | 0.306 | 2.900 |
| 4 | 0.301 | 2.929 |
| 5 | 0.316 | 2.826 |
| 6 | 0.312 | 3.109 |
| 7 | 0.323 | 2.853 |
| 8 | 0.302 | 3.050 |
| 9 | 0.324 | 3.020 |
| 10 | 0.310 | 2.887 |
| M | 0.311 | 2.950 |
| SD | 0.008 | 0.095 |
| CV | 2.5% | 3.2% |
Precision testing result statistical form between table 5, analysis
4th, the rate of recovery：
Cortisol sample A of the concentration in the range of 5 μ g/L～6 μ g/L is added to concentration in 0.08 μ g/L～0.2 μ g/L
In the range of sample B in, volume ratio between the sample A for being added and sample B is 1:9, calculated back according to equation below (2)
Yield R, as a result should meet rate of recovery 90%-110%, and test data is as shown in table 6 below.
In formula：The R-- rate of recovery；
The volume of V-- sample A；
V0The volume of sample B；
C samples B adds the detectable concentration after sample A；
c0The detectable concentration of sample B；
csThe concentration of sample A.
Table 6, rate of recovery testing result statistical form
5. clinical testing：
The present embodiment reagent compares 500 parts of clinical serum pattern detection results with external authority's reagent (Abbott Laboratories' reagent), surveys
Test result totally meets and reaches 99.9%, it is seen that kit prepared by the present embodiment method has preferable consistent with hospital measured value
Property.Wherein 100 parts clinical test results are enumerated, as shown in table 7 below.
Table 7, clinical test results statistical form
In sum, a kind of ANS salt that the present invention is provided is combined the kit of releasing agent and detection Blood cortisol, this
Invent the cortex that the cortisol of combining form in blood of human body sample is changed into free form by the compound releasing agent of ANS salt
Alcohol, and the content of free cortisol is detected by chemiluminescent new technique.From the compound releasing agent of ANS salt can from different
Glucocorticosteroid albumen combines, and can fully discharge the cortisol in blood with reference to state；Described chemiluminescence
For acridinium ester chemiluminescent, acridinium ester chemiluminescent is luminous for flash type, few during illuminating, can completely produced by catching reaction
Photon；Acridinium ester is artificial synthetic, and the anti-interference to sample interfering material is higher, using acridinium ester label detection trip
Content from cortisol is very sensitive, makes the assay of total cortisol in blood sample more accurate.The present invention has operation
Easy, sensitivity is high, verification and measurement ratio is fast, low cost, be easy to the advantages of automating.
It should be appreciated that the application of the present invention is not limited to above-mentioned citing, and for those of ordinary skills, can
To be improved according to the above description or be converted, all these modifications and variations should all belong to the guarantor of claims of the present invention
Shield scope.
Claims (10)
1. a kind of detection Blood cortisol kit, it is characterised in that including following components：The magnetic of coated cortisol antibody
The compound releasing agent of pearl, the cortisol BSA compounds of acridinium ester label, ANS salt and cortisol calibration object.
2. the kit of detection Blood cortisol according to claim 1, it is characterised in that also include luminous cleaning fluid
A and luminous cleaning fluid B.
3. according to claim 1 detection Blood cortisol kit, it is characterised in that the magnetic bead surface bag
It is wrapped with any one in carboxyl, epoxy radicals, toluene Huang acyl group.
4. according to claim 1 detection Blood cortisol kit, it is characterised in that acridinium ester and cortisol
The molar ratio of the compound substance markers of BSA is between 1:2.5-1:Between 10.
5. according to claim 1 detection Blood cortisol kit, it is characterised in that the acridinium ester label
Cortisol BSA compounds are Cortisol-3-BSA compounds.
6. according to claim 1 detection Blood cortisol kit, it is characterised in that the cortisol calibration object
Cortisol antigen for the alcohol hormone serum-dilution that decorticates.
7. according to claim 2 detection Blood cortisol kit, it is characterised in that the luminous cleaning fluid A
Including following components：The disodium hydrogen phosphate of 50-100g/L, the sodium dihydrogen phosphate of 5-30g/L, the sodium chloride of 50-200g/L,
The peroxidating of liquid bio preservative Proclin-300,0.5%-5% of Tween-20,0.1%-10% of 0.1%-10%
Hydrogen and the nitric acid of 1-2M.
8. according to claim 2 detection Blood cortisol kit, it is characterised in that the luminous cleaning fluid B
Including following components：The disodium hydrogen phosphate of 50-100g/L, the sodium dihydrogen phosphate of 5-30g/L, the sodium chloride of 50-200g/L,
The hydroxide of liquid bio preservative Proclin-300,0.01-2.5M of Tween-20,0.1%-10% of 0.1%-10%
Sodium and 0.1%-2%TritonX-100.
9. a kind of ANS salt is combined releasing agent, it is characterised in that the compound releasing agent of the ANS salt includes following components：3-15mg/
Ml ANS salt, 0.1-0.5%NaN3, 100-200mM NaCl and 20-60mM barbital sodiums.
10. ANS salt according to claim 9 is combined releasing agent, it is characterised in that the ANS salt has following structure：
Above-mentioned R+For NH4 +、K+Or Na+.
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| CN107290526A (en) * | 2017-08-24 | 2017-10-24 | 太原瑞盛生物科技有限公司 | A kind of chemiluminescence detection kit of tetracycline and preparation method thereof |
| CN108982835A (en) * | 2018-05-31 | 2018-12-11 | 湖南远璟生物技术有限公司 | A kind of estradiol magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof |
| CN109438539A (en) * | 2018-10-25 | 2019-03-08 | 美康生物科技股份有限公司 | Aldosterone derivative contains its chemiluminescence immune detection reagent kit and preparation method thereof |
| CN109444408A (en) * | 2018-10-26 | 2019-03-08 | 成都普利泰生物科技有限公司 | A kind of kit detecting animal blood cortisol |
| CN110579615A (en) * | 2019-09-06 | 2019-12-17 | 广州科方生物技术股份有限公司 | Release agent universal for steroid hormone in serum and preparation method thereof |
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| CN110579615A (en) * | 2019-09-06 | 2019-12-17 | 广州科方生物技术股份有限公司 | Release agent universal for steroid hormone in serum and preparation method thereof |
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