CN106645695A - Chemiluminescent immunoassay kit for anti-zona-pellucida antibody and preparation method of chemiluminescent immunoassay kit - Google Patents
Chemiluminescent immunoassay kit for anti-zona-pellucida antibody and preparation method of chemiluminescent immunoassay kit Download PDFInfo
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- CN106645695A CN106645695A CN201610503871.4A CN201610503871A CN106645695A CN 106645695 A CN106645695 A CN 106645695A CN 201610503871 A CN201610503871 A CN 201610503871A CN 106645695 A CN106645695 A CN 106645695A
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- membrane antibody
- vitellary membrane
- vitellary
- detection reagent
- reagent kit
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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
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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
- G01N33/531—Production of immunochemical test materials
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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
- G01N33/54346—Nanoparticles
Abstract
The invention discloses a chemiluminescent immunoassay kit for an anti-zona-pellucida antibody and a preparation method of the chemiluminescent immunoassay kit. The chemiluminescent immunoassay kit for the anti-zona-pellucida antibody comprises carboxylation magnetic microparticles enveloped with anti-zona-pellucida recombinant proteins as well as a chemiluminescent marker marked by anti-human immunoglobulin. The chemiluminescent immunoassay kit for the anti-zona-pellucida antibody can adopt a full-automatic chemiluminescent immunity analyzer as a detection tool to finish detection of the anti-zona-pellucida antibody. Experiments prove that the detection sensitivity of the chemiluminescent immunoassay kit for the anti-zona-pellucida antibody reaches 1 U/L, which is increased at least ten times as compared with the sensitivity of the traditional anti-zona-pellucida antibody detection method, and the detection precision of the chemiluminescent immunoassay kit for the anti-zona-pellucida antibody is higher.
Description
Technical field
The present invention relates to vitro detection field, more particularly to a kind of anti-vitellary membrane antibody chemiluminescence immune detection reagent kit
And preparation method thereof.
Background technology
Oolemma is one layer surrounds the non-cellular analyte gelatin sample acidoglycoprotein film of cytula before egg mother cell and implantation, main
Specific sperm acceptor is included by what 3 kinds of glycoprotein were constituted, its effect is induction sperm acrosome reaction, smart ovum identification, knot
Close, many inseminations are penetrated and prevent, under normal physiological conditions, after sperm is combined with oolemma, by the enzyme system of sperm
The dissolution of local is produced, after fertilization oolemma recovers integrality, protects the development of embryonated egg.
Anti- vitellary membrane antibody is combined in vivo with oolemma, sperm ovum binding can be prevented, between interference ovum and follicle cell
Reaction, cause egg cell and ovum locking.On the other hand anti-vitellary membrane antibody declines can the locomitivity of sperm, affect essence
It is sub through Ovulation prediction and up, and affect capacitation；Can also damaging action be produced to the cytula of oolemma, as a result lead in addition
Cause is unable to normal development and arrives and miscarry.Research both at home and abroad finds higher vitellary membrane antibody recall rate in infertile women.Cause
This anti-vitellary membrane antibody detection is also significant with the infertile patient for the treatment of to clinical diagnosis.
At present the common methods of the anti-vitellary membrane antibody of clinical detection have enzyme linked immunosorbent assay, enzyme-catalyzed chemical luminescence method, but
These methods all have some shortcomings part.
First, enzyme linked immunosorbent assay
Enzyme linked immunosorbent assay (ELISA) is widely used, but the method there is also following weak points：
(1) using 12 × 8 types, 6 × 8 types, 8 × 12 types or the hole Special micro porous plate of complete plate 96 as antigen coat apparatus and anti-
Container is answered, 12 batches, 6 batches, 8 batches or whole plate first use can only be divided into when in use, it is impossible to carried out independent, single
The detection of person-portion；
(2) reagent type used by quantitative determining is more, and each detection reagent will be contained with reagent bottle, and often be made
It is required for changing imbibition nozzle to be filled into respectively in the micropore of microwell plate during with a kind of reagent, not only reagent bottle species is more, filling
The operation of reagent is also extremely loaded down with trivial details；
(3) lack the corresponding mark to detection information, can only just will appreciate that by checking the mark of kit external packing box or know
The product batch number and term of validity information of detection reagent are known, and the information known is uncontrolled in detection process, with very big
Randomness；
(4) detection reagent easily causes the cross pollution between various reagents and shadow in open space in detection process
Ring the accuracy of testing result；
(5) dosage more than detection process using manual operations, reagent or sample is not bery accurate, and operating process is extremely loaded down with trivial details and multiple
It is miscellaneous, bust is susceptible to, the degree of accuracy of testing result and precision are poor；
(6) item number × 48/96 person-portion is in the quantity configuration of detection project reagent set and using on, if necessary to examine
10 projects are surveyed, then the configuration of reagent and the use of number must be 10 × 48/96 person-portions, if only a sample needs detection 10
Individual different project, it is also desirable to configure the reagent of 10 × 48/96 person-portions, haves the shortcomings that inadequate economical rationality.
2nd, chemoluminescence method
Chemoluminescence method can be divided into direct chemiluminescence and enzyme-catalyzed chemical luminescence by principle of luminosity.
Enzyme-catalyzed chemical luminescence mainly has horseradish peroxidase（HRP）With two kinds of alkaline phosphatase, but there is certain office
Sex-limited, horseradish peroxidase major defect is：Luminol, also can be by H2O2 in the case of existing without horseradish peroxidase
Oxidation itself lights, and background is of a relatively high, affects signal to noise ratio, and kinetics is complicated, and influence factor is more, is as a result not sufficiently stable,
The substrate for obtaining sensitivity height and plateau length is not easy.Alkaline phosphatase major defect is：Substrate reach plateau when
Between long, substrate high cost, cause testing cost high, patient burden's weight.
Acridinium ester compares enzyme-catalyzed chemical luminescence and has detailed advantage, main performance as the direct chemiluminescence of label
：Reaction does not need catalyst, as long as alkaline environment can be carried out, is swift in response, and background luminescence is low, and signal to noise ratio is high, disturb because
Element is few, and reagent stability is good, can be simple with two-point calibration, system, exciting liquid low cost, acridinium ester easily and protein bind, and
Photon yield is not reduced after connection.
The content of the invention
Based on this, it is necessary to provide a kind of detection sensitivity higher anti-vitellary membrane antibody chemiluminescence immunoassay detection reagent
Box and preparation method thereof.
A kind of anti-vitellary membrane antibody chemiluminescence immune detection reagent kit, including：Anti- vitellary membrane antibody recombinant protein coating
Carboxylated magnetic particle and human immunoglobulins mark chemiluminescent labels.
In one embodiment, it is described anti-in the magnetic particle of the coated carboxylated of the anti-vitellary membrane antibody recombinant protein
The ratio of the magnetic particle of vitellary membrane antibody recombinant protein and the carboxylated is 1：25~35.
In one embodiment, in the chemiluminescent labels of human immunoglobulins' mark, the anti-human immunity
The ratio of globulin and the chemiluminescent labels is 50：1~10.
In one embodiment, the particle diameter of the magnetic particle of the carboxylated is 0.05 μm ~ 1 μm.
In one embodiment, the chemiluminescent labels are luminol, different luminol, tris (bipyridine) ruthenium or acridine
Ester.
In one embodiment, also including Chemoluminescent substrate, the Chemoluminescent substrate includes A liquid and B liquid.
In one embodiment, the A liquid is H2O2Solution, the B liquid is NaOH solution.
In one embodiment, also product are calibrated including anti-vitellary membrane antibody.
In one embodiment, the anti-vitellary membrane antibody calibration product for concentration be respectively 1U/L, 10U/L, 100U/L,
The solution of the anti-vitellary membrane antibody of 500U/L, 1000U/L and 2000U/L.
A kind of preparation method of above-mentioned anti-vitellary membrane antibody chemiluminescence immune detection reagent kit, comprises the steps：
The suspension of the magnetic particle of carboxylated is taken, Magneto separate goes after supernatant to use MES buffer solutions resuspended, is subsequently added into the EDC aqueous solution,
The surface carboxyl groups of the magnetic particle of activated carboxyl, are subsequently added into anti-vitellary membrane antibody recombinant protein, suspension 2h ~ 10h, magnetic under room temperature
Separating use Tris buffer solutions resuspended after removal supernatant, obtains the magnetic particle of the coated carboxylated of anti-vitellary membrane antibody recombinant protein；
And
Human immunoglobulins are taken, is added and mixed after carbonate buffer solution, be subsequently adding after chemiluminescent labels and mix, room temperature
Removal of impurities after lower lucifuge reaction 1h ~ 2h, obtains the chemiluminescent labels of human immunoglobulins' mark.
This anti-vitellary membrane antibody chemiluminescence immune detection reagent kit can be with Full-automatic chemiluminescence immunoassay analysis meter
To detect instrument, this anti-vitellary membrane antibody chemiluminescence immune detection reagent kit of detection of anti-vitellary membrane antibody is completed, passed through
Experiment, its detection sensitivity reaches 1U/L, and the detection method sensitivity relative to traditional anti-vitellary membrane antibody at least improves
10 times, the accuracy of detection of this anti-vitellary membrane antibody chemiluminescence immune detection reagent kit is higher.
Description of the drawings
Fig. 1 is the flow process of the preparation method of the anti-vitellary membrane antibody chemiluminescence immune detection reagent kit of an embodiment
Figure；
Fig. 2 is the anti-vitellary membrane antibody canonical plotting that embodiment 3 is obtained.
Specific embodiment
It is understandable to enable the above objects, features and advantages of the present invention to become apparent from, it is below in conjunction with the accompanying drawings and concrete real
Apply example to be described in detail the specific embodiment of the present invention.Elaborate many details in the following description in order to
Fully understand the present invention.But the present invention can be implemented with being much different from alternate manner described here, art technology
Personnel can do similar improvement in the case of without prejudice to intension of the present invention, therefore the present invention is not embodied as by following public
Restriction.
The anti-vitellary membrane antibody chemiluminescence immune detection reagent kit of one embodiment, including：Anti- vitellary membrane antibody restructuring
The chemiluminescent labels of the magnetic particle of the coated carboxylated of albumen and human immunoglobulins' mark.
Preferably, in the magnetic particle of the coated carboxylated of anti-vitellary membrane antibody recombinant protein, anti-vitellary membrane antibody restructuring egg
It is in vain 1 with the ratio of the magnetic particle of carboxylated：25~35.
Preferably, in the chemiluminescent labels of human immunoglobulins' mark, anti-human immunoglobulins send out with chemistry
The ratio of signal thing is 50：1~10.
Preferably, the particle diameter of the magnetic particle of carboxylated is 0.05 μm ~ 1 μm.
Chemiluminescent labels can be luminol, different luminol, tris (bipyridine) ruthenium or acridinium ester.Wherein, chemiluminescence
Label is preferably acridinium ester.
In other examples, above-mentioned anti-vitellary membrane antibody chemiluminescence immune detection reagent kit also includes chemiluminescence
Substrate solution.
Chemoluminescent substrate includes A liquid and B liquid.A liquid can be H2O2Solution, B liquid can be NaOH solution.
In the present embodiment, A liquid is the H that concentration is 0.1mol/L2O2Solution, B liquid is that concentration is molten for the NaOH of 0.25mol/L
Liquid.
In other examples, above-mentioned anti-vitellary membrane antibody chemiluminescence immune detection reagent kit also includes anti-oolemma
Antibody calibrates product.
Anti- vitellary membrane antibody calibration product for concentration be respectively 1U/L, 10U/L, 100U/L, 500U/L, 1000U/L and
The solution of the anti-vitellary membrane antibody of 2000U/L.
Specifically, anti-vitellary membrane antibody can be configured to concentration by anti-vitellary membrane antibody calibration product using standard items buffer solution
The respectively solution of the anti-vitellary membrane antibody of 1U/L, 10U/L, 100U/L, 500U/L, 1000U/L and 2000U/L.
When this anti-vitellary membrane antibody chemiluminescence immune detection reagent kit is used for the detection of anti-vitellary membrane antibody, using entirely certainly
Dynamic chemical illumination immunity analysis instrument antagonism vitellary membrane antibody calibration product are detected that drafting calibration curve is built in computer software；
Then actual sample is tested, concentration of specimens is calculated according to sample luminous value；Finally resist vitellary membrane antibody Full-automatic chemiluminescence
Immunoassay system carries out performance（Sensitivity, linear, precision, interference）Evaluation.
This anti-vitellary membrane antibody chemiluminescence immune detection reagent kit can be with Full-automatic chemiluminescence immunoassay analysis meter
To detect instrument, this anti-vitellary membrane antibody chemiluminescence immune detection reagent kit of detection of anti-vitellary membrane antibody is completed, passed through
Experiment, its detection sensitivity reaches 1U/L, and the detection method sensitivity relative to traditional anti-vitellary membrane antibody at least improves
10 times, the accuracy of detection of this anti-vitellary membrane antibody chemiluminescence immune detection reagent kit is higher.
Additionally, this anti-vitellary membrane antibody chemiluminescence immune detection reagent kit has further the advantage that：
1st, acridinium ester being selected as marker material, and being applied to chemiluminescence immunoassay system, the luminescence system is directly change
Learn luminous, compared with traditional enzyme-catalyzed chemical luminescence, the reaction does not need the participation of enzyme, more cost-effective；
2nd, from the chemiluminescence immunoassay system range of linearity width of acridinium ester, 1U/L ~ 1000U/L can be reached, and it is traditional
The inspection range of linearity of the detection method of anti-vitellary membrane antibody is 20U/L ~ 1000U/L；
3rd, acridinium ester chemiluminescent immunoassay system repeatability is high, and within 5%, this is other chemistry to batch interior and difference between batch
Luminescence immunoassay system is unapproachable；
4th, chemiluminescence immunoassay system has realized the quantitative of sample, by built-in calibration curve to test software, only needs to survey
Sample originally can directly obtain the concentration value of sample；
5th, chemiluminescence immunoassay system can realize that the addition of full-automation, reagent and sample has instrument to complete entirely, operation
It is easier, reduce artificial error.
The preparation method of above-mentioned anti-vitellary membrane antibody chemiluminescence immune detection reagent kit as shown in Figure 1, including it is as follows
Step：
The suspension of the magnetic particle of carboxylated is taken, Magneto separate goes after supernatant to use MES buffer solutions resuspended, is subsequently added into the EDC aqueous solution,
The surface carboxyl groups of the magnetic particle of activated carboxyl, are subsequently added into anti-vitellary membrane antibody recombinant protein, suspension 2h ~ 10h, magnetic under room temperature
Separating use Tris buffer solutions resuspended after removal supernatant, obtains the magnetic particle of the coated carboxylated of anti-vitellary membrane antibody recombinant protein.
MES（2- (N- morpholines) ethyl sulfonic acid）The concentration of buffer solution is 0.02M, and pH is 5.5.
The concentration of Tris buffer solutions is 0.1M and contains 2%BSA, and pH is 8.0.
EDC（1- ethyl -3- (3- dimethyl aminopropyls)-carbodiimides）The concentration of the aqueous solution is 10mg/mL ~ 20mg/
The ratio of the magnetic particle of mL, EDC and carboxylated is 0.05：0.1~1.
Preferably, in the magnetic particle of the coated carboxylated of anti-vitellary membrane antibody recombinant protein, anti-vitellary membrane antibody restructuring egg
It is in vain 1 with the ratio of the magnetic particle of carboxylated：25~35.
Preferably, the particle diameter of the magnetic particle of carboxylated is 0.05 μm ~ 1 μm.
Human immunoglobulins are taken, are added and mixed after carbonate buffer solution, be subsequently adding after chemiluminescent labels and mix,
Removal of impurities after lucifuge reaction 1h ~ 2h, obtains the chemiluminescent labels of human immunoglobulins' mark under room temperature.
Carbonate buffer solution concentration is 0.1M, and pH is 9.0 ~ 9.5,
The operation of removal of impurities is centrifugation desalting column desalination, and concrete operations are：First respectively with pure water and TBS buffer solutions（40 mM
Tris-HCl, 0.5% BSA, 1% NaCl, pH 8.0）Centrifugation desalting column is processed, the anti-vitellary membrane antibody weight for obtaining is eventually adding
The solution of the magnetic particle of the coated carboxylated of histone, finally collects the liquid in centrifuge tube.
Preferably, human immunoglobulins mark chemiluminescent labels in, anti-vitellary membrane antibody recombinant protein with change
The ratio for learning luminous marker is 50：1~10.
Chemiluminescent labels can be luminol, different luminol, tris (bipyridine) ruthenium or acridinium ester.Wherein, chemiluminescence
Label is preferably acridinium ester.
What the magnetic particle of the coated carboxylated of anti-vitellary membrane antibody recombinant protein for obtaining and human immunoglobulins marked
Chemiluminescent labels combination is obtained above-mentioned anti-vitellary membrane antibody chemiluminescence immune detection reagent kit.
This anti-vitellary membrane antibody chemiluminescence immune detection reagent kit is when in use, in addition it is also necessary to Chemoluminescent substrate and
Anti- vitellary membrane antibody calibrates product.
Chemoluminescent substrate and anti-vitellary membrane antibody calibration product can voluntarily be prepared and obtained.
Chemoluminescent substrate includes A liquid and B liquid.A liquid can be H2O2Solution, B liquid can be NaOH solution.
In the present embodiment, A liquid is the H that concentration is 0.1mol/L2O2Solution, B liquid is that concentration is molten for the NaOH of 0.25mol/L
Liquid.
Specifically, anti-vitellary membrane antibody can be configured to concentration by anti-vitellary membrane antibody calibration product using standard items buffer solution
The respectively solution of the anti-vitellary membrane antibody of 1U/L, 10U/L, 100U/L, 500U/L, 1000U/L and 2000U/L.
The preparation method of this anti-vitellary membrane antibody chemiluminescence immune detection reagent kit is simple and convenient, obtained anti-transparent
Detection sensitivity with antibody chemical luminescence immunity detection reagent is higher, has a good application prospect.
It is below specific embodiment.
Embodiment 1：The preparation of anti-vitellary membrane antibody chemiluminescence immune detection reagent kit
（1）The preparation of the magnetic particle of the coated carboxylated of anti-vitellary membrane antibody recombinant protein：
Take the magnetic particle for 0.05 μm ~ 1 μm of carboxylated containing 50mg particle diameters（MagnaBind21353）Suspension, Magneto separate goes
Supernatant, uses 0.02 M, pH to be that 5.5 MES buffer solutions are resuspended, adds the EDC aqueous solution of the 10mg/mL of the new configurations of 1mL, activates magnetic
Bead surface carboxyl, adds the anti-vitellary membrane antibody recombinant proteins of 4mg（Biorbyt, article No. orb48780）, be suspended 6h under room temperature, magnetic
Separate, remove supernatant, with the 0.1M containing 2%BSA, pH is that 8.0 Tris buffer solutions are resuspended to 1mg/mL, obtain anti-oolemma and resist
The magnetic particle of the coated carboxylated of body weight histone, every bottle of 5mL packing be stored in 4 DEG C it is standby.
（2）The preparation of the acridinium ester of human immunoglobulins' mark：
The human immunoglobulins that 50 μ L concentration are 25mg/mL are taken, the carbonic acid that 150 μ L concentration are that 0.1M, pH are 9.0 ~ 9.5 is added
Salt buffer, mixes, and is subsequently adding the acridine ester solution that 1.5 μ L concentration are 5mg/mL and mixes, lucifuge reaction under room temperature, after 1.5h
Take out, desalting column desalting processing is centrifuged with the zeba of 2mL, carried out with pure water and TBS buffer solutions respectively first in desalination processes
Process, be eventually adding the acridine ester solution of the human immunoglobulins' mark for obtaining, collect the liquid in centrifuge tube to preserving pipe
Obtain the acridinium ester of human immunoglobulins' mark, every bottle of 5mL packing be stored in 4 DEG C it is standby.
（3）Anti- vitellary membrane antibody calibrates the preparation of product：
Use standard items buffer solution（40 mM Tris-HCl, 0.5% BSA, 1% NaCl, pH 8.0）By the configuration of anti-vitellary membrane antibody
It is 0U/L, 10U/L, 100U/L, 500U/L, 1000U/L and 2000U/L into concentration, per bottle of 0.5 mL packing is lyophilized, 4 DEG C of preservations
It is standby.
Embodiment 2：Anti- vitellary membrane antibody chemical luminous immune detection method
With Full-automatic chemiluminescence immunoassay analysis meter（YHLO, article No. iFlash3000）To detect instrument, between methodology pattern is
Connect immunization, i.e. instrument and sequentially add the sample of 50 μ L, the coated carboxylated of anti-vitellary membrane antibody recombinant protein of 50 μ L
The anti-vitellary membrane antibody treatment fluid of magnetic particle and 50 μ L, after 20 min of reaction, then the human immunoglobulins' a word used for translation for adding 50 μ L
Pyridine ester, after 20 min of reaction, carries out Magneto separate, and reactant mixture is sent into darkroom by instrument, sequentially adds luminous substrate A liquid
（H2O2）And B liquid（NaOH）Luminescence-producing reaction is carried out, luminous value is finally recorded.
Embodiment 3：Anti- vitellary membrane antibody chemiluminescence immune detection reagent kit performance evaluation
Detected using the method antagonism vitellary membrane antibody calibration product in embodiment 2, obtain drawing calibration curve such as Fig. 2 institutes
Show.
Then to then testing actual sample, concentration of specimens is calculated according to sample luminous value.
The detection of sensitivity：
With reference to CLSI EP17-A file recommendation experimental programs, anti-vitellary membrane antibody chemiluminescence immune detection reagent kit is calculated
Sensitivity, the sensitivity tried to achieve is 1U/L.
Linear detection：
Linear analysis is done for 1U/L, 10U/L, 100U/L, 500U/L, 1000U/L and 2000U/L standard items to concentration, line is calculated
Property coefficient correlation, r=0.9996, in addition, the range of linearity of kit antagonism vitellary membrane antibody sample detection be 1U/L ~
1000U/L。
Precision is determined：
Concentration is taken for 50U/L and the anti-vitellary membrane antibody samples of 500U/L two, each sample each concentration respectively do 3 it is parallel, use
Three batches of kits are detected, calculated in kit batch and difference between batch, as a result shown in the kit batch and difference between batch is respectively less than
5%。
Interference is tested：
Taking pooled serum and adding chaff interference respectively includes：Combined with bilirubin, unconjugated bilirubin, hemoglobin, ascorbic acid, glycerine
Ester, adding proportion is according to 1：20 are carried out, and are determined pooled serum respectively and be with the addition of the measured value of pooled serum after various chaff interferences,
Deviation therebetween is calculated, with ± 10% as tolerance interval.As a result show, interference reaches the files-designated of NCCLS
Standard, can be used for the accurate evaluation of the anti-vitellary membrane antibody situation of clinical labororatory.
The contrast experiment of embodiment 4, anti-vitellary membrane antibody chemiluminescence immune detection reagent kit
It is respectively the anti-vitellary membrane antibody of 0,50U/L to concentration with chemical luminescence detection method and traditional enzyme linked immunosorbent assay
Sample detects that two methods detection sensitivity is compared, and data are as shown in the table：
As can be seen from the above table, the sensitivity of chemical luminescence detection method improves more than 10 times compared with enzyme linked immunosorbent assay.
Embodiment described above only expresses the several embodiments of the present invention, and its description is more concrete and detailed, but can not
Therefore it is interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art,
Without departing from the inventive concept of the premise, some deformations and improvement can also be made, these belong to the protection model of the present invention
Enclose.Therefore, the protection domain of patent of the present invention should be defined by claims.
Claims (10)
1. a kind of anti-vitellary membrane antibody chemiluminescence immune detection reagent kit, it is characterised in that include：Anti- vitellary membrane antibody restructuring
The chemiluminescent labels of the magnetic particle of the coated carboxylated of albumen and human immunoglobulins' mark.
2. anti-vitellary membrane antibody chemiluminescence immune detection reagent kit according to claim 1, it is characterised in that described anti-
In the magnetic particle of the coated carboxylated of vitellary membrane antibody recombinant protein, the anti-vitellary membrane antibody recombinant protein and the carboxylated
Magnetic particle ratio be 1：25~35.
3. anti-vitellary membrane antibody chemiluminescence immune detection reagent kit according to claim 1, it is characterised in that described anti-
In the chemiluminescent labels of human immunoglobulin(HIg) mark, the anti-vitellary membrane antibody recombinant protein and the chemiluminescent labeling
The ratio of thing is 50：1~10.
4. anti-vitellary membrane antibody chemiluminescence immune detection reagent kit according to claim 1, it is characterised in that the carboxylic
The particle diameter of the magnetic particle of base is 0.05 μm ~ 1 μm.
5. anti-vitellary membrane antibody chemiluminescence immune detection reagent kit according to claim 1, it is characterised in that describedization
Luminous marker is luminol, different luminol, tris (bipyridine) ruthenium or acridinium ester.
6. anti-vitellary membrane antibody chemiluminescence immune detection reagent kit according to claim 1, it is characterised in that also include
Chemoluminescent substrate, the Chemoluminescent substrate includes A liquid and B liquid.
7. anti-vitellary membrane antibody chemiluminescence immune detection reagent kit according to claim 6, it is characterised in that the A
Liquid is H2O2Solution, the B liquid is NaOH solution.
8. anti-vitellary membrane antibody chemiluminescence immune detection reagent kit according to claim 1, it is characterised in that also include
Anti- vitellary membrane antibody calibrates product.
9. anti-vitellary membrane antibody chemiluminescence immune detection reagent kit according to claim 8, it is characterised in that described anti-
Vitellary membrane antibody calibration product are respectively the anti-transparent of 1U/L, 10U/L, 100U/L, 500U/L, 1000U/L and 2000U/L for concentration
Solution with antibody.
10. anti-vitellary membrane antibody chemiluminescence immune detection reagent kit of the one kind according to any one of claim 1 ~ 9
Preparation method, it is characterised in that comprise the steps：
The suspension of the magnetic particle of carboxylated is taken, Magneto separate goes after supernatant to use MES buffer solutions resuspended, is subsequently added into the EDC aqueous solution,
The surface carboxyl groups of the magnetic particle of activated carboxyl, are subsequently added into anti-vitellary membrane antibody recombinant protein, suspension 2h ~ 10h, magnetic under room temperature
Separating use Tris buffer solutions resuspended after removal supernatant, obtains the magnetic particle of the coated carboxylated of anti-vitellary membrane antibody recombinant protein；
And human immunoglobulins are taken, and add and mixed after carbonate buffer solution, it is subsequently adding after chemiluminescent labels and mixes, room temperature
Removal of impurities after lower lucifuge reaction 1h ~ 2h, obtains the chemiluminescent labels of human immunoglobulins' mark.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1423129A (en) * | 2001-12-07 | 2003-06-11 | 暨南大学 | Kit for detecting egg vitellary membrane antibody |
| CN101504416A (en) * | 2008-05-20 | 2009-08-12 | 湖南工业大学 | Novel methods for detecting bacillus coli by gold-coating magnetic granule in-situ initiating high-sensibility chemical luminescence |
-
2016
- 2016-06-30 CN CN201610503871.4A patent/CN106645695A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1423129A (en) * | 2001-12-07 | 2003-06-11 | 暨南大学 | Kit for detecting egg vitellary membrane antibody |
| CN101504416A (en) * | 2008-05-20 | 2009-08-12 | 湖南工业大学 | Novel methods for detecting bacillus coli by gold-coating magnetic granule in-situ initiating high-sensibility chemical luminescence |
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Application publication date: 20170510 |