CN109001451A - GlyFn chemical luminescent analysis reagent kid and the purposes in the screening of preeclampsia early stage - Google Patents
GlyFn chemical luminescent analysis reagent kid and the purposes in the screening of preeclampsia early stage Download PDFInfo
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- CN109001451A CN109001451A CN201810576085.6A CN201810576085A CN109001451A CN 109001451 A CN109001451 A CN 109001451A CN 201810576085 A CN201810576085 A CN 201810576085A CN 109001451 A CN109001451 A CN 109001451A
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- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 49
- 201000011461 pre-eclampsia Diseases 0.000 title claims abstract description 49
- 238000012216 screening Methods 0.000 title claims abstract description 33
- 239000000126 substance Substances 0.000 title claims abstract description 31
- 239000011324 bead Substances 0.000 claims abstract description 29
- 239000000758 substrate Substances 0.000 claims abstract description 24
- 238000001514 detection method Methods 0.000 claims abstract description 18
- RXNXLAHQOVLMIE-UHFFFAOYSA-N phenyl 10-methylacridin-10-ium-9-carboxylate Chemical compound C12=CC=CC=C2[N+](C)=C2C=CC=CC2=C1C(=O)OC1=CC=CC=C1 RXNXLAHQOVLMIE-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000012360 testing method Methods 0.000 claims abstract description 13
- 239000003550 marker Substances 0.000 claims abstract description 10
- 210000002966 serum Anatomy 0.000 claims abstract description 8
- 108010017384 Blood Proteins Proteins 0.000 claims abstract description 7
- 102000004506 Blood Proteins Human genes 0.000 claims abstract description 7
- 239000011229 interlayer Substances 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 24
- 239000011248 coating agent Substances 0.000 claims description 13
- 238000000576 coating method Methods 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 12
- 239000007853 buffer solution Substances 0.000 claims description 11
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 4
- 229940098773 bovine serum albumin Drugs 0.000 claims description 4
- 208000002296 eclampsia Diseases 0.000 claims description 3
- 210000004369 blood Anatomy 0.000 claims description 2
- 239000008280 blood Substances 0.000 claims description 2
- 230000003760 hair shine Effects 0.000 claims description 2
- 206010015037 epilepsy Diseases 0.000 claims 1
- 238000003745 diagnosis Methods 0.000 abstract description 6
- 230000001900 immune effect Effects 0.000 abstract description 5
- 238000004140 cleaning Methods 0.000 description 8
- 230000035935 pregnancy Effects 0.000 description 8
- 238000003908 quality control method Methods 0.000 description 8
- 238000005406 washing Methods 0.000 description 8
- 230000009514 concussion Effects 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- 206010020772 Hypertension Diseases 0.000 description 5
- 108010082093 Placenta Growth Factor Proteins 0.000 description 5
- 102100035194 Placenta growth factor Human genes 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 201000010099 disease Diseases 0.000 description 4
- 239000006210 lotion Substances 0.000 description 4
- 239000011541 reaction mixture Substances 0.000 description 4
- 239000012898 sample dilution Substances 0.000 description 4
- 206010001580 Albuminuria Diseases 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 235000013601 eggs Nutrition 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- DZBUGLKDJFMEHC-UHFFFAOYSA-N acridine Chemical compound C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 230000035487 diastolic blood pressure Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000009434 installation Methods 0.000 description 2
- 210000002826 placenta Anatomy 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 1
- 108010082126 Alanine transaminase Proteins 0.000 description 1
- 102100037362 Fibronectin Human genes 0.000 description 1
- 108010067306 Fibronectins Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- -1 Pyridine ester Chemical class 0.000 description 1
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 1
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 1
- 101900125653 Vascular endothelial growth factor receptor 1 (isoform 2) Proteins 0.000 description 1
- 102300040083 Vascular endothelial growth factor receptor 1 isoform 2 Human genes 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 208000015294 blood coagulation disease Diseases 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000008753 endothelial function Effects 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 210000004700 fetal blood Anatomy 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000004789 organ system Anatomy 0.000 description 1
- 230000004796 pathophysiological change Effects 0.000 description 1
- 230000009984 peri-natal effect Effects 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 201000001474 proteinuria Diseases 0.000 description 1
- JUJWROOIHBZHMG-UHFFFAOYSA-N pyridine Substances C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000035488 systolic blood pressure Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 229940116269 uric acid Drugs 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
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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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/36—Gynecology or obstetrics
- G01N2800/368—Pregnancy complicated by disease or abnormalities of pregnancy, e.g. preeclampsia, preterm labour
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
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- Biotechnology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
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- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Gynecology & Obstetrics (AREA)
- Pregnancy & Childbirth (AREA)
- Reproductive Health (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Early screening preeclampsia is carried out using GlyFn plasma protein as marker, pregnant 9-12 weeks sample can be detected.GlyFn chemical luminescent analysis reagent kid provided by the invention is used for early screening preeclampsia.GlyFn chemical luminescent analysis reagent kid uses two step double-antibody sandwich immunologic detection methods, GlyFn in test serum is in conjunction with anti-GlyFn antibody coated on magnetic bead, again in conjunction with the anti-GlyFn antibody of acridinium ester label, it forms magnetic bead and is coated with object-GlyFn- acridinium ester label interlayer type immune complex, luminous value is detected after substrate is added, the concentration of GlyFn in sample is calculated by standard curve.The sensibility and specificity of this kit test result diagnosis preeclampsia respectively reaches 97%, 93%, is a kind of detection kit of function admirable, can carry out the screening of preeclampsia early stage.
Description
Technical field
The present invention relates to preeclampsia screening technical fields, try more particularly to a kind of GlyFn chemiluminescence quantitative detection
Agent box and purposes.
Background technique
There is hypertension, albuminuria after gestation 20 weeks in pregnant preceding normotensive pregnant woman, claims preeclampsia, or for first
Million eclampsias are one of five kinds of situations of hypertensive disorder in pregnancy, send out disease for gestational period spy, can influence each organ system of body
System.According to statistics, the disease incidence of external pregnant woman's preeclampsia is 5%~12%, China 9.4%.Preeclampsia can lead to puerpera
And perinatal infants dead rate, the pregnant woman of preeclampsia is died of every year in global range is up to 50,000 to 7.5 ten thousand.
The diagnosis basis of preeclampsia is two indexs of pregnant woman's blood pressure and Urine proteins at present: 1. gestation are shunk after 20 weeks
Pressure >=140mmHg and (or) diastolic pressure >=90mmHg；2. with albuminuria >=0.3g/24 hours or Random urine protein >=(+).But
This two index sensibility and specificities are low, hypertension occur without albuminuria or lasting egg during some women gestation
Albiduria does not have hypertension but corresponding clinical symptoms occurs；And after there is hypertension and proteinuria, disease is quickly grown.Cause
This this two index can not Accurate Prediction which pregnant woman preeclampsia can occur and how to be in progress after the onset.
In recent years there are many emerging biomarkers, including blood plasma GAP-associated protein GAP A (PPAP-A), fetal blood red eggs
White, fibronectin (Fn), placenta growth factor (PLGF), soluble vascular endothelial growth factor receptor1 (sFlt1) etc., wherein
PLGF/sFlt1 is the product applied in the market, and the NPV that joint-detection diagnoses preeclampsia is 99.3%, PPV 36.7%.
Screening in the prior art needs to judge by multiple indexs, cannot be with single Indexs measure, more seriously
Pregnant early diagnosis is not can be carried out.Because marker in the prior art is related to bad endothelial function, inflammatory reaction, blood coagulation disorders etc.
Pathophysiological change relevant to preeclampsia, concentration level has been second trimester when changing in maternal blood circulation.Therefore,
How screening preeclampsia is carried out in early days, become a big technical problem of this field.
Therefore, in view of the shortcomings of the prior art, provide a kind of GlyFn chemical luminescent analysis reagent kid and application thereof with gram
It is very necessary to take prior art deficiency.
Summary of the invention
A kind of GlyFn chemiluminescence quantitative detection is provided it is an object of the invention to avoid the deficiencies in the prior art place
Kit and test method,
There is provided GlyFn can be realized the pregnant preeclampsia early stage screening of single index progress.
The object of the present invention is achieved by the following technical measures.
Purposes of the plasma protein as marker in early screening preeclampsia.Preferably, refer in early days pregnant 9-12 weeks.
Present invention simultaneously provides a kind of GlyFn chemical luminescent analysis reagent kids, are used for early screening preeclampsia.
Preferably, above-mentioned GlyFn chemical luminescent analysis reagent kid, contains:
R1, abbreviation magnetic bead are coated with object, the 0.01M PBS buffer solution containing the magnetic bead coating object for being coated with anti-GlyFn antibody；
R2, abbreviation acridinium ester label, the 0.01M PBS buffer solution of the anti-GlyFn antibody containing acridinium ester label.
Preferably, above-mentioned GlyFn chemical luminescent analysis reagent kid, containing the magnetic bead coating for being coated with anti-GlyFn antibody
The pH value of the 0.01M PBS buffer solution of object be 7.2, include 0.1% polysorbas20 (v/v), 5% bovine serum albumin(BSA) (w/v),
0.5%Proclin300 (v/v).
Preferably, above-mentioned GlyFn chemical luminescent analysis reagent kid, the anti-GlyFn antibody containing acridinium ester label
The pH value of 0.01M PBS buffer solution is 7.2, includes 0.1% polysorbas20 (v/v), 5% bovine serum albumin(BSA) (w/v), 0.5%
Proclin300(v/v)。
Preferably, above-mentioned GlyFn chemical luminescent analysis reagent kid, also contains:
Substrate solution A: contain H2O2Aqueous solution, as preexciting liquid；
Substrate solution B: the aqueous solution containing NaOH, as exciting liquid.
The present invention provides the use that early screening preeclampsia is used for according to above-mentioned GlyFn chemical luminescent analysis reagent kid
On the way.
Preferably, the method for the screening of preeclampsia early stage being carried out by above-mentioned GlyFn chemical luminescent analysis reagent kid,
GlyFn chemical luminescent analysis reagent kid uses two step double-antibody sandwich immunologic detection methods, the GlyFn in test serum with
Coated anti-GlyFn antibody combines on magnetic bead, then in conjunction with the anti-GlyFn antibody of acridinium ester label, forms magnetic bead and is coated with object-
GlyFn- acridinium ester label interlayer type immune complex detects luminous value after substrate is added, is calculated by standard curve
The concentration of GlyFn in sample.
Preferably, the method for above-mentioned progress preeclampsia early stage screening diagnoses the sensibility and specificity point of preeclampsia
Do not reach 97%, 93%.
The present invention carries out early screening preeclampsia using GlyFn plasma protein as marker, can be to pregnant 9-12 weeks
Sample is detected.GlyFn chemical luminescent analysis reagent kid provided by the invention is used for early screening preeclampsia.
GlyFn chemical luminescent analysis reagent kid uses two step double-antibody sandwich immunologic detection methods, the GlyFn in test serum with
Coated anti-GlyFn antibody combines on magnetic bead, then in conjunction with the anti-GlyFn antibody of acridinium ester label, forms magnetic bead and is coated with object-
GlyFn- acridinium ester label interlayer type immune complex detects luminous value after substrate is added, is calculated by standard curve
The concentration of GlyFn in sample.The sensibility and specificity of this kit test result diagnosis preeclampsia respectively reaches 97%,
93%, it is a kind of detection kit of function admirable, the screening of preeclampsia early stage can be carried out.
Specific embodiment
The invention will be further described with the following Examples.
Embodiment 1.
Purposes of the GlyFn plasma protein as marker in early screening preeclampsia is provided.Pregnant 9- can be achieved in early stage
12 weeks time.
GlyFn can be used for First Trimester (pregnant week 9 weeks) and diagnose preeclampsia, the predictable serious bad clinic of GlyFn
The generation of final result；GlyFn can guiding treatment mode select, instruct whether terminal pregnancy.
Test discovery, 100 μ g/mL of every raising, prediction pregnant time reduce by 4 days (P < 0.01)；Systolic pressure increases 1.39mm
Hg (P=0.04)；Diastolic pressure increases 1.14mm Hg (P=0.01)；Neonatal weight reduces 129.4g；Uric acid increases 13.6 μ
Mol/L (P < 0.01), glutamic-pyruvic transaminase increase 5.88U/L (P < 0.01)；The pregnant age and egg of GlyFn level and diagnosis preeclampsia
Albiduria is unrelated.Test also found that the GlyFn water average specific of the early, middle and late three period placenta in preeclampsia of gestation is normally organized significantly
It increases, test also found that placenta in preeclampsia disease degree is heavier, and average GlyFn level increase weekly is more.Therefore it can be with
GlyFn plasma protein realizes purposes in early screening preeclampsia as marker.
With sFlt1, PlGF, sFlt1/PlGF index is compared, and GlyFn predicts that the accuracy of preeclampsia is best.It is right with its
The sensibility and specificity for the kit test result diagnosis preeclampsia answered respectively reaches 97%, 93%, in terms of disease incidence 5%
It calculates, it is a kind of detection kit of function admirable that negative predictive value and positive predictive value, which are respectively 47%, 89%,.
Embodiment 2.
A kind of GlyFn chemical luminescent analysis reagent kid is used for early screening preeclampsia, contains:
R1, abbreviation magnetic bead are coated with object, the 0.01M PBS buffer solution containing the magnetic bead coating object for being coated with anti-GlyFn antibody；
GlyFn chemical luminescent analysis reagent kid, the PH of the 0.01MPBS buffer containing the magnetic bead coating object for being coated with anti-GlyFn antibody
Value is 7.2, includes 0.1% polysorbas20 (v/v), 5% bovine serum albumin(BSA) (w/v), 0.5%Proclin300 (v/v)；
R2, abbreviation acridinium ester label, the 0.01M PBS buffer solution of the anti-GlyFn antibody containing acridinium ester label.Containing acridine
The pH value of the 0.01M PBS buffer solution of the anti-GlyFn antibody of ester label is 7.2, includes 0.1% polysorbas20 (v/v), 5% ox blood
Pure albumen (w/v), 0.5%Proclin300 (v/v)；
Substrate solution A: contain H2O2Aqueous solution, as preexciting liquid；
Substrate solution B: the aqueous solution containing NaOH, as exciting liquid.
The method for carrying out the screening of preeclampsia early stage by the GlyFn chemical luminescent analysis reagent kid, GlyFn chemistry
The immue quantitative detection reagent box that shines uses two step double-antibody sandwich immunologic detection methods, wraps on the GlyFn and magnetic bead in test serum
The anti-GlyFn antibody of quilt combines, then in conjunction with the anti-GlyFn antibody of acridinium ester label, forms magnetic bead and is coated with object-GlyFn- a word used for translation
Pyridine ester marker interlayer type immune complex detects luminous value after substrate is added, is calculated in sample by standard curve
The concentration of GlyFn.
The method for carrying out the screening of preeclampsia early stage by the GlyFn chemical luminescent analysis reagent kid diagnoses eclampsia
The sensibility and specificity of early period respectively reaches 97%, 93%.
Embodiment 3.
The screening of preeclampsia early stage, concrete operations are carried out by the GlyFn chemical luminescent analysis reagent kid of embodiment 2
Process is as follows:
1. kit, in use, before installation, needs magnetic bead coating object (R1) being gently turned upside-down about 30 for the first time
It is secondary, so that bead particulates is uniformly dispersed.It needs not continue to mix after loading magnetic bead coating object (R1) for the first time.
2. magnetic bead coating object (R1) and acridinium ester label (R2) are installed in reagent rack.It is chosen in instrumentation interface
Reagent position scans the two dimensional code in reagent rack, reagent rack is put into agent bin.
3. preparing GlyFn calibration object and GlyFn quality-control product by GlyFn calibration object specification and GlyFn quality-control product specification.
4. preparing Sample dilution by Sample dilution specification.
5. preparing cleaning solution by concentrated cleaning solution specification.
6. preparing substrate solution A and substrate solution B by substrate solution specification.
7. calibration
Curve is clicked, GlyFn project is chosen, two-dimensional barcode information subsidiary in kit is scanned, curve will be automatically generated.It will
GlyFn calibration object is put on sample rack, is pushed into sample rack, and the editing sample information on operation interface chooses GlyFn project, often
A calibration object does 2 multiple holes, and sample type is set as " CA ", clicks " RUN " after determining.
8. detection
Sample is mixed well using forward horizontal stand to room temperature.
GlyFn quality-control product and sample are put on sample rack (sample size should be greater than 300 μ L), sample rack is pushed into, is operating
Editing sample information on interface chooses GlyFn project, clicks " RUN " after determining.System will perform the following operations (calibration operation
It is identical):
Total incubation time 15 minutes.
● determinand (calibration object, quality-control product, sample) is sent to entry point.
● reaction cup is loaded into operation channel.
● 30 μ L determinands are drawn respectively to reaction cup.
● reaction cup is transported to reagent position in storehouse, and 50 μ L reagent R1 are added.
● after concussion mixing, reaction cup is transported to and is incubated for storehouse, 37 DEG C are incubated for 10 minutes.
● reaction cup is transported to washing channel, Magneto separate is carried out, cleans reaction mixture with washing lotion, repeats magnetic point
From-cleaning 4 times.
● reaction cup is transported again to reagent position in storehouse, and 50 μ L reagent R2 are added.
● after concussion mixing, reaction cup is transported to and is incubated for storehouse, 37 DEG C are incubated for 5 minutes.
● reaction cup is transported to washing channel, Magneto separate is carried out, cleans reaction mixture with washing lotion, repeats magnetic point
From-cleaning 4 times.
● reaction cup is transported to substrate channels, 100 μ L substrate solution A, concussion mixing is added.
● reaction cup is transported to sense channel, to storehouse is detected 100 μ L substrate solution B are added, and examine immediately in crawl reaction cup
Luminous signal is surveyed, GlyFn concentration is calculated.
● crawl reaction cup to waste bin.
9. result judgement
This project reagent generates a mark using four parameter Logistic curve-fit data reduction methods (4PLC, Y are weighted)
Directrix curve.
Embodiment 4.
For detecting 5 normal pregnancies serum, 5 Cases with Preeclampsia pregnancy serums, illustrate kit operation of the invention
Process and Effect of screening.
1. needing for magnetic bead coating object (R1) to be gently turned upside-down about 30 times, bead particulates being made to be uniformly dispersed before installation.
It needs not continue to mix after loading magnetic bead coating object (R1) for the first time.
2. magnetic bead coating object (R1) and acridinium ester label (R2) are installed in reagent rack.It is chosen in instrumentation interface
Reagent position scans the two dimensional code in reagent rack, reagent rack is put into agent bin.
3. preparing GlyFn calibration object and GlyFn quality-control product by GlyFn calibration object specification and GlyFn quality-control product specification.
4. preparing Sample dilution by Sample dilution specification.
5. preparing cleaning solution by concentrated cleaning solution specification.
6. preparing substrate solution A and substrate solution B by substrate solution specification.
7. calibration
Curve is clicked, GlyFn project is chosen, two-dimensional barcode information subsidiary in kit is scanned, curve will be automatically generated.It will
GlyFn calibration object is put on sample rack, is pushed into sample rack, and the editing sample information on operation interface chooses GlyFn project, often
A calibration object does 2 multiple holes, and sample type is set as " CA ", clicks " RUN " after determining.
8. detection
Sample is mixed well using forward horizontal stand to room temperature.
GlyFn quality-control product and sample are put on sample rack, push-in sample rack, the editing sample information in operation interface,
GlyFn project is chosen, clicks " RUN " after determining.System will perform the following operations:
Total incubation time 15 minutes.
● 20 parts of samples (calibration object, quality-control product, sample) to be measured are sent to entry point.
● reaction cup is loaded into operation channel.
● 30 μ L determinands are drawn respectively to reaction cup.
● reaction cup is transported to reagent position in storehouse, and 50 μ L reagent R1 are added.
● after concussion mixing, reaction cup is transported to and is incubated for storehouse, 37 DEG C are incubated for 10 minutes.
● reaction cup is transported to washing channel, Magneto separate is carried out, cleans reaction mixture with washing lotion, repeats magnetic point
From-cleaning 4 times.
● reaction cup is transported again to reagent position in storehouse, and 50 μ L reagent R2 are added.
● after concussion mixing, reaction cup is transported to and is incubated for storehouse, 37 DEG C are incubated for 5 minutes.
● reaction cup is transported to washing channel, Magneto separate is carried out, cleans reaction mixture with washing lotion, repeats magnetic point
From-cleaning 4 times.
● reaction cup is transported to substrate channels, 100 μ L substrate solution A, concussion mixing is added.
● reaction cup is transported to sense channel, 100 μ L substrate solution B are added to storehouse is detected in crawl reaction cup,
And luminous signal is detected immediately, calculate GlyFn concentration.
● crawl reaction cup to waste bin.
10. result judgement
A standard curve, detection are generated using four parameter Logistic curve-fit data reduction methods (4PLC, Y are weighted)
The results are shown in Table 1.
Table 1
The present invention carries out early screening preeclampsia using GlyFn plasma protein as marker, can be to pregnant 9-12 weeks
Sample is detected.GlyFn chemical luminescent analysis reagent kid provided by the invention is used for early screening preeclampsia.
GlyFn chemical luminescent analysis reagent kid uses two step double-antibody sandwich immunologic detection methods, the GlyFn in test serum with
Coated anti-GlyFn antibody combines on magnetic bead, then in conjunction with the anti-GlyFn antibody of acridinium ester label, forms magnetic bead and is coated with object-
GlyFn- acridinium ester label interlayer type immune complex detects luminous value after substrate is added, is calculated by standard curve
The concentration of GlyFn in sample.The sensibility and specificity of this kit test result diagnosis preeclampsia respectively reaches 97%,
93%, it is a kind of detection kit of function admirable, the screening of preeclampsia early stage can be carried out.
Finally it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should be managed
Solution, can with modification or equivalent replacement of the technical solution of the present invention are made, without departing from technical solution of the present invention essence and
Range.
Claims (10)
- Purposes of the 1.GlyFn albumen as marker in early screening preeclampsia.
- 2. purposes of the GlyFn plasma protein according to claim 1 as marker in early screening preeclampsia, It is characterized in that, refers in early days pregnant 9-12 weeks.
- 3. a kind of GlyFn chemical luminescent analysis reagent kid, which is characterized in that be used for early screening preeclampsia.
- 4. GlyFn chemical luminescent analysis reagent kid according to claim 3, which is characterized in that contain:R1, abbreviation magnetic bead are coated with object, the 0.01M PBS buffer solution containing the magnetic bead coating object for being coated with anti-GlyFn antibody；R2, abbreviation acridinium ester label, the 0.01M PBS buffer solution of the anti-GlyFn antibody containing acridinium ester label.
- 5. GlyFn chemical luminescent analysis reagent kid according to claim 4, which is characterized in that containing the anti-GlyFn of coating The pH value of the 0.01M PBS buffer solution of the magnetic bead coating object of antibody is 7.2, and it is pure to include 0.1% polysorbas20 (v/v), 5% ox blood Albumen (w/v), 0.5%Proclin300 (v/v).
- 6. GlyFn chemical luminescent analysis reagent kid according to claim 5, which is characterized in that contain acridinium ester label Anti- GlyFn antibody 0.01M PBS buffer solution pH value be 7.2, include 0.1% polysorbas20 (v/v), 5% bovine serum albumin White (w/v), 0.5%Proclin300 (v/v).
- 7. GlyFn chemical luminescent analysis reagent kid according to claim 6, which is characterized in that also contain:Substrate solution A: contain H2O2Aqueous solution, as preexciting liquid；Substrate solution B: the aqueous solution containing NaOH, as exciting liquid.
- 8. according to GlyFn chemical luminescent analysis reagent kid described in claim 3-7 any one for early screening The purposes of epilepsy early period.
- 9. before carrying out early stage eclampsia by GlyFn chemical luminescent analysis reagent kid described in claim 3-7 any one The method of phase screening, which is characterized in that GlyFn chemical luminescent analysis reagent kid uses two step double-antibody sandwich immune detections Method, the GlyFn in test serum is in conjunction with anti-GlyFn antibody coated on magnetic bead, then the anti-GlyFn with acridinium ester label Antibody combines, and forms magnetic bead and is coated with object-GlyFn- acridinium ester label interlayer type immune complex, and detection shines after substrate is added Value, the concentration of GlyFn in sample is calculated by standard curve.
- 10. the side that GlyFn chemical luminescent analysis reagent kid according to claim 8 carries out the screening of preeclampsia early stage Method, which is characterized in that the sensibility and specificity for diagnosing preeclampsia respectively reaches 97%, 93%.
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