CN110031634A - A kind of detection kit and its application method of Human plactnta growth factor - Google Patents
A kind of detection kit and its application method of Human plactnta growth factor Download PDFInfo
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- CN110031634A CN110031634A CN201910332813.3A CN201910332813A CN110031634A CN 110031634 A CN110031634 A CN 110031634A CN 201910332813 A CN201910332813 A CN 201910332813A CN 110031634 A CN110031634 A CN 110031634A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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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
- G01N33/6854—Immunoglobulins
Abstract
The invention discloses a kind of detection kits of Human plactnta growth factor.The main of the kit includes specificity capture antibody and specific detection antibody；Specificity capture antibody and specific detection antibody are selected from following antibody: goat polyclonal antibodies 655170, mouse monoclonal antibody 655565, goat polyclonal antibodies 446, and specificity capture antibody and the Species origin of specific detection antibody pairing answer difference；Kit of the invention further includes solid phase carrier, cleaning solution, enhancement solution, marker, calibration object and test buffer.The present invention also provides the application methods of the detection kit.Detection kit of the present invention can be used for detecting Human plactnta growth factor, with easy to operate, time-consuming short, the advantages that accuracy is good, high sensitivity, the range of linearity are wide, strong interference immunity, good stability has good potential applicability in clinical practice in terms of the auxiliary screening of Tang Shi and Disadvantage pregnancy reaction.
Description
Technical field
The invention belongs to biochemical fields, are related to a kind of kit, and in particular to a kind of time-resolved fluoroimmunoassay
Detect the kit and its application method of Human plactnta growth factor.
Background technique
Placenta growth factor (placental growth factor, PlGF) most earlier than 1991 by Maglione etc. from
It isolates and purifies and obtains in the placenta cdna library of people.PlGF is a kind of secretory homodimer glycoprotein, the assignment of genes gene mapping in
14q24-q31.According to the different montage mode of encoding gene RNA, there are 4 kinds of isomers: i.e. PlGF-1, PlGF-2, PlGF- at present
3, PlGF-4, base sequence and vascular endothelial growth factor (vascular endothelial growth factor,
VEGF) gene has the homology of height, belongs to VEGF family, with the fms sample junket ammonia for being located at trophocyte and vascular endothelial cell
Acid kinase receptor -1 (fms-like tyrosine kianse-1, Flt-1), which is conducted after combining by transmembrane signal, plays pathology
Physiological role.PlGF is mainly synthesized by placenta syncytiotrophoblast, in the internal organs such as the heart, lung, thyroid gland, skeletal muscle and blood vessel
The cells such as endothelial cell, vascular smooth muscle cells, the bone marrow cell of source property also have expressed.PlGF is in normal pregnancy placenta and mother
In high expression in blood, and male and normal unpregnancy women are nearly no detectable PlGF.PlGF can promote trophocyte when early pregnancy to increase
It grows and breaks up, due to its significant angiogenesis characteristic, so that the factor and gestation, inflammatory reaction, cardiovascular disease, tumour etc.
Suffer from close relationship.PlGF is abundant to be expressed in placenta, related to angiogenesis, endothelial cell and trophocyte function, right
Placentation and development of fetus important role, abnormal expression and Averse pregnancy outcomes and pregnant related complication are close
It is related.The variation of PlGF level is peak shape during gestation, and when 7~15 pregnant week, content is lower (average about 40~50pg/mL), 28~
Peak (about 180pg/mL) when 30 pregnant week, and when 39~41 pregnant week is reduced to 55~65pg/mL, this supply oxygen with placenta variation and
PLGF physiological function is related.Research shows that PlGF and ectopic pregnancy, threatened abortion, intrauterine growth restriction, pre-eclampsia, Tang
A variety of Averse pregnancy outcomes such as Cotard are related:
Preeclampsia (pre-eclampsia, PE) is the gestational period peculiar disease, is to lead to pregnant and lying-in women and peri-natal infant perinatal period
The major reason of illness and death, it is clinical with gestation emerging hypertension (systolic pressure > 140mmHg or diastole later in 20 weeks
Press > 90mmHg), albuminuria (in > 0.3g/24 hour) be main feature.The disease incidence of PE is 2%~8%, 10%~15%
Pregnant and lying-in women's cause of the death be PE and eclampsia.According to statistics, the disease incidence of China's preeclampsia is about 9.5%.Therefore, preeclampsia is pre-
It surveys and primary prevention seems of crucial importance.The PE cause of disease is complicated, and etiology and pathogenesis not yet illustrates completely, currently, existing big
Quantity research proves the change of sFlt-1 and PlGF concentration obviously earlier than Attack of Preeclampsia.There is clinical research to show within 2004
SFlt-1 concentration just increases for about 5 weeks before Attack of Preeclampsia, and about 9~11 weeks just before Attack of Preeclampsia for PlGF concentration
It begins to decline, starts sharply to decline before morbidity 5 weeks, concentration when 1 week before the onset concentration has been very close to morbidity；Early onset, evening
Are there is symptom in sFlt-1, PlGF concentration and sFlt-1/PlGF of hair style and non-premature labor type preeclampsia high risk pregnant woman
It is preceding that dynamic change occurs.
The screening of Tang Shi has been popularized, but screening mode more commonly used at present, either early stage or mid-term, single
For pure serological screening when false positive rate is 5%, recall rate only has 60%~70% or so.Show PlGF pregnant according to the study
In the Prenatal Screening of early stage Down syndrome abnormal pregnancy, false positive can reduce, and improve recall rate.Cowans NJ etc. recognizes
For pregnant 11~13+6The PlGF in week can be with pregnant woman age, pregnancy-associated plasma protein, free β-human chorionic gonadtropin
It is listed in the marker of 21- three-body detection together with nuchal translucency.Donalson K etc. has studied identical vacation as the result is shown
Under positive rate, if PlGF is added in screening mode, recall rate can be improved 4%~7%, and under identical recall rate, add
Enter PlGF, can almost reduce the false positive rate of half.If alpha-fetoprotein and PlGF are added simultaneously, recall rate be can be improved
5%~8%.Down ratio after the ratio that Wald NJ etc. has studied PlGF decline is higher than 14 weeks when mid-term, and and mid-term
It compares, the screening method that PlGF is added in early stage can significantly reduce the false positive rate of detection, and the effect does not have then when mid-term
Have so obvious.
Since content is lower in mother's serum in pregnant early stage by PlGF, it is detected as pg grades of levels, averagely about 40 when 7~15 pregnant week
~50pg/mL, and mother's gestation hypertension, fetus pre-eclampsia, nourish Down's syndrome fetus etc., PlGF contains in female serum
It measures obviously relatively low compared with normal pregnancies.12~15 pregnant week PlGF < 32pg/mL, 16~20 20 pregnant weeks of pregnant week PlGF < 60pg/mL, >
When PlGF < 100pg/mL, prompts placental function abnormal, there is preeclampsia risk.When any pregnant week PlGF < 12pg/mL, prompt
Placental function has huge damage, need to tightly monitor.Extraneous panimmunity reaction factor is (when injection volume, reaction temperature, reaction
Between, vibration frequency etc.) will affect the median of PlGF, therefore detect PlGF and need the reagent of a kind of high sensitivity and stable
Reaction system.
Therefore, serological index of the PlGF as a very potential prediction Averse pregnancy outcomes, has important
Social and economic implications.Existing PlGF detection kit product type is ELISA detection kit, is taken a long time, and model is detected
Enclose relatively narrow, sensitivity and stability not enough, are affected by environment and operator, are unable to satisfy wanting for clinical diagnosis
It asks, therefore needs to develop a kind of PlGF detection kit with clinical value.
Summary of the invention
In view of the above-mentioned problems, the present invention provide it is a kind of for detecting the detection kit (time resolution of placenta growth factor
Fluorescence analysis), after advanced optimizing, there is the advantages that time-consuming shorter, detection range is wider, and high sensitivity, stability are good,
With good clinical value.
To achieve the above object, the technical scheme adopted by the invention is as follows:
A kind of detection kit of Human plactnta growth factor, including specificity capture antibody and specific detection antibody；
The specificity capture antibody and the specific detection antibody are selected from following antibody: goat polyclonal antibodies 655170, mouse
Monoclonal antibody 655565, goat polyclonal antibodies 446, and the kind of specificity capture antibody and specific detection antibody pairing
Answer difference in source；The mouse monoclonal antibody 655565 uses amino acid sequence for Ala21-Arg149 (Genbank Shelf number
For the Bacillus coli expression recombined human PlGF of P49763), it is immunized obtained by mouse；The goat polyclonal antibodies 655170 and goat
Polyclonal antibody 446 is to use amino acid sequence for the large intestine bar of Ala21-Arg149 (Genbank Shelf number is CAA38698)
Bacterium expresses recombined human PlGF, immune goat gained.
The pairing of mouse monoclonal antibody 655565 and goat polyclonal antibodies 655170 and mouse monoclonal antibody
655565 with the pairings of goat polyclonal antibodies 446, the calibration object that Human plactnta growth factor sterling is prepared, pregnant early screening
Preferable match reaction is all presented in the normal sample of (early sieve) and the normal sample of second trimester screening (middle sieve), indicates that this is anti-
Body is to preferable potential applicability in clinical practice.
The detection kit further include: solid phase carrier, marker, calibration object, cleaning solution, enhancement solution, and experiment
Buffer；The specificity capture antibody or Streptavidin are coated on solid phase carrier；The marker is rare earth element, institute
It states marker and the specific detection antibody or specific detection antibody marker or Streptavidin is made in Streptavidin
Marker；The calibration object is that the calibrated product dilution of people's recombinant placenta growth factor is diluted through concentration gradient.
Concentration containing Human plactnta growth factor sterling in each calibration object is respectively 0,20,100,500,2500,
10500pg/mL is lyophilized after each calibration object packing and saves.
Preferably, the rare earth element is europium.
Preferably, the calibration object dilution is 50mmol/L, trishydroxymethylaminomethane-hydrochloride buffer of pH 7.8
Liquid, containing 5% bovine serum albumin(BSA), the thimerosal of 20ppm and 0.01% Sodium azide.
Preferably, the cleaning solution is the sodium chloride of 1.125g/L, the polysorbas20 of 0.02% (v/v), 30ppmThe mixture of the tris-HCI buffer composition of 300 preservatives.
Preferably, the enhancement solution is the mixture that Qula is led to, glacial acetic acid, chelating agent, purified water form.
Preferably, it is described further include test buffer be by buffer, preservative, immune signal reinforcing agent, protein and
The mixed solution of chelating agent composition: the buffer is 50mmol/L, trishydroxymethylaminomethane-hydrochloride buffer of pH 7.8
Liquid；The preservative is to contain 0.02% (v/v's)300, the thimerosal of 20ppm；The chelating agent is 0.001%
(v/v) disodium ethylene diamine tetraacetate；The immune signal reinforcing agent is 5%~20% (v/v) SignalBoostTMImmune letter
One or both of number reinforcing agent, 0.05%~0.2% (m/v) Macrogol 6000；The protein is 0.1% (v/v)
Mice serum, the calf serum of 10% (v/v), γ-ox globulin of 0.5% (m/v) and 5% (m/v) bovine serum albumin
It is one or more of white.
The chelating agent can remove the metal ion for influencing reaction；Albumen in the immune signal reinforcing agent can be reduced instead
Non-specific binding during answering, SignalBoostTMImmune signal reinforcing agent can reduce signal-to-noise ratio, accelerate antigen-antibody
Reaction, and the detection success rate of low-abundance protein can be effectively improved, reduce the usage amount of detection antibody marker, polyethylene glycol
6000 can improve the reactivity of antigen and antibody.
The application method of the detection kit are as follows: solid phase biological element Avidin method or bridge-type biotin-labeled pentylamine method or
Sandwich method, in which:
The solid phase biological element Avidin method, steps are as follows:
A) the specificity capture antibody response of biotin labeling is added in solid-phase coating Streptavidin,
B) it is directly added into sample to be reacted, the reaction of specific detection antibody marker is added after washing, or add after washing
Enter sample to be reacted, the reaction of specific detection antibody marker is added after washing, or sample and specificity are directly added simultaneously
The reaction of antibody marker is detected, or sample and the reaction of specific detection antibody marker is added after washing simultaneously,
C) enhancement solution is added after washing, tests and analyzes；
The bridge-type biotin-labeled pentylamine method, steps are as follows:
A) solid-phase coating specificity captures antibody, and sample is added and is reacted,
B) the specific detection antibody reaction of biotin labeling is added after washing, marked by streptavidin object is added after washing
Reaction,
Or biotin labeling specific detection antibody, the reaction of marked by streptavidin object is added after washing simultaneously, or directly
The reaction of biotin labeling specific detection antibody is added, the reaction of marked by streptavidin object is added after washing,
Or biotin labeling specific detection antibody, the reaction of marked by streptavidin object are directly added simultaneously,
C) enhancement solution is added after washing, tests and analyzes；
The sandwich method, steps are as follows:
A) solid-phase coating specificity captures antibody,
B) sample is added to be reacted, the reaction of specific detection antibody marker is added after washing, or sample is added simultaneously
It is reacted with specific detection antibody marker,
C) enhancement solution is added after washing, tests and analyzes.
Preferably, the application method of the detection kit uses sandwich method, and steps are as follows: solid-phase coating is special
Opposite sex capture antibody, is added sample and is reacted, washed, and the reaction of specific detection antibody marker is added, is added and increases after washing
Strong liquid tests and analyzes.
The application method of detection kit according to the present invention, four kinds of modes of solid phase biological element Avidin method are vulnerable to the external world
The interference of biotin, technique and complicated for operation, the cost is relatively high, compared with other reaction patterns, in detection sensitivity not
See and be obviously improved there is apparent HOOK effect in latter two, and detection sensitivity is low.Bridge-type biotin-labeled pentylamine method it is several
Kind of mode fails to play the role of amplified signal, technique and complicated for operation, and the cost is relatively high, and promotion is had no in detection sensitivity,
Three kinds due to steric effect afterwards, in conjunction with insecure, be easy to make to be immunoreacted desorption or cause to wash away in board-washing process anti-
Compound is answered, causes sensitivity to decline instead, and interference of the latter two vulnerable to extraneous biotin, there are apparent HOOK effects
It answers.The first technique and operation relative ease of sandwich method, advantage of lower cost, most with solid phase biological element Avidin method
Good detection sensitivity and the range of linearity is suitable, and not by the interference of extraneous biotin, but second there are apparent HOOK effect,
Detection sensitivity is low.Therefore the first method of preferably sandwich method, also referred to as double-antibody sandwich two step method.
Specifically, the step of carrying out the detection of Human plactnta growth factor using the detection kit is as follows:
(1) solid-phase coating: specificity capture antibody is dissolved in coating buffer and captures antibody at containing 3~7 μ g/mL
Coating buffer, take 100~200 μ L coating buffer volumes carry out solid phase carrier coating；
(2) it is loaded: taking 50~150 μ L of test specimens to be separately added into each solid phase carrier (being coated with specificity capture antibody) anti-
Ying Weizhong；
(3) it is incubated for: the solid phase carrier equipped with test specimens being carried out incubation 30~120 minutes on shaker, frequency of oscillation
For 750~1150rpm, incubation temperature is 20~30 DEG C；
(4) board-washing: test specimens are discarded, and wash each reaction position 1~6 with cleaning solution working solution (26 times of cleaning solution dilution)
It is secondary；
(5) antibody marker working solution (the specificity inspection of 50~150 μ L antibody incubation: is separately added into each reaction position
It surveys antibody marker and test buffer is formulated by 1:1200, ready-to-use), and 30~120 points are incubated on shaker
Clock, frequency of oscillation are 750~1150rpm, and incubation temperature is 20~30 DEG C；
(6) board-washing: marker working solution is discarded, and washs each reactant 2~10 times with cleaning solution；
(7) 50~150 μ L enhancement solutions, and 1~30 point of oscillating reactions signal enhancing: is added into each reaction position respectively
Clock；
(8) it fluorescence signal detection and analysis: is detected using time resolution immunofluorescence assay instrument, and according to testing number
According to progress interpretation of result.
Specifically, the process of specificity capture antibody coating solid phase carrier is as follows: taking suitable antibody to be added slow to coating
In fliud flushing dilution, coating buffer is added and carries out solid-phase coating, discards coating buffer, and wash solid phase 1 time, is then carried out again with confining liquid
Closing, gets rid of and dries, and is vacuum-packed, 2~8 DEG C save backup；Cleaning solution, the confining liquid of solid phase washing are conventional reagent.
Wherein, the coating buffer is any one of following buffer: the phosphate-buffered of 20mmol/L, pH 4.5
Liquid, 50mmol/L, carbonic acid buffer, the 50mmol/L of pH 9.6, the tris-HCI buffer of pH 7.8,
The citrate buffer of 50mmol/L, pH 4.5.
Preferably, the coating buffer volume is 150 μ L, and capture antibody concentration described in the coating buffer is 5 μ g/mL, institute
Stating coating buffer is 20mmol/L, the phosphate buffer of pH 4.5.
Preferably, the test specimens are serum or blood plasma.
Preferably, coating buffer volume described in step (1) is 150 μ L, and capture antibody concentration described in the coating buffer is 5
μ g/mL, the coating buffer are 20mmol/L, the phosphate buffer of pH 4.5；The additional amount of test specimens is in step (2)
100μL；Incubation temperature during step (3), (5) are incubated for is 30 DEG C, incubation time 60min, frequency of oscillation 1150rpm；
Board-washing number is 2 times in step (4)；Board-washing number in step (6) is 6 times；The additional amount of enhancement solution is 100 μ in step (7)
L, reaction time are 5 minutes.
The present invention has clinical value, hyperergic antibody pair by screening, and to the reaction system of detection
Such as peridium concentration, coating volume, injection volume, reaction time have carried out serial optimization, establish a set of double-antibody sandwich two
Based on footwork, take time-resolved fluoroimmunoassay as the Human plactnta growth factor detection architecture of final analysis means, and prepares
At commercially available detection kit.The detection kit operating process is simple, has compared to existing kit time-consuming short, sensitive
The advantages that degree is high, the range of linearity is wide, precision is good, strong interference immunity, can precisely reflect PlGF content in sample, in Tang Shi
Auxiliary screening and prediction etc. of Disadvantage pregnancy reaction there is huge potential applicability in clinical practice.
Detailed description of the invention
Fig. 1 is different dissociation-enhancing duration of oscillation calibration object actual measurement fluorescence count value CPs result.
Fig. 2 is the dose-response curve that time-resolved fluoroimmunoassay of the present invention detects placenta growth factor.
Specific embodiment
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with the drawings and specific embodiments pair
The present invention is described further.
Embodiment 1
A kind of component of time-resolved fluoroimmunoassay detection placenta growth factor reagent includes:
1) micro reaction plate: being coated with capture antibody, and peridium concentration is 5 μ g/mL；
2) calibration object: calibration object contains 6 A, B, C, D, E, F concentration points, and calibration object A is zero-dose, calibration object B, C, D,
E, F contains people's recombinant placenta growth factor of series of concentrations gradient.
3) antibody marker is detected: the detection antibody containing europium label placenta growth factor.
4) test buffer: contain 0.02% in the tris-HCI buffer of 50mmol/L, pH 7.8
(v/v)300, the SignalBoost of the thimerosal of 20ppm, 10% (v/v)TMImmune signal reinforcing agent, 0.1%
(m/v) disodium ethylene diamine tetraacetate of the calf serum of PEG6 000,10% (v/v), 0.001% (m/v).
5) it is concentrated washing lotion: the Tween-20 of sodium chloride, 0.2mL/L containing 1.125g/L, 30ppm300 is anti-
The tris-HCI buffer of rotten agent.
6) enhancement solution: logical, glacial acetic acid, chelating agent, purified water containing Qula.
1. reagent prepares
1) micro reaction plate: reagent and required amount of micro reaction plate are balanced to room temperature (20~25 DEG C).It is remaining
Micro reaction plate be placed in time valve bag it is closed and in 2~8 DEG C save.
2) cleaning liquid: cleaning solution and purified water are added in clean container by 1:25 and mixed, as cleaning
Liquid is spare.
3) it detects antibody marker working solution: being prepared using in preceding 30min, measure as needed, will test antibody marker
It adds in clean disposable container and mixes by 1:1 200 with test buffer；When secondary experiment is finished.
2. detecting step
1) 100 μ L calibration objects are pipetted with pipettor and sample sequentially adds in the reacting hole of micro reaction plate.
2) micro reaction plate at room temperature, oscillation incubation 90min.
3) it after the first step is incubated for, is washed 2 times with cleaning liquid.
4) detection 100 μ L of antibody marker working solution is sequentially added in the reacting hole of micro reaction plate.
5) at 30 DEG C, oscillation incubation 60min.
6) it after second step is incubated for, is washed 6 times with cleaning liquid.
7) 100 μ L of enhancement solution is sequentially added into each reacting hole.
8) solid phase carrier detects after vibrating 5min at room temperature, completes to detect and analyzed in 30min.
Embodiment 2
The reagent component of embodiment 2 is identical with embodiment 1, and reagent prepares and detection process is as follows:
1. reagent prepares
1) micro reaction plate: reagent and required amount of micro reaction plate are balanced to room temperature (20~25 DEG C).It is remaining
Micro reaction plate be placed in time valve bag it is closed and in 2~8 DEG C save.
2) cleaning liquid: cleaning solution and purified water are added in clean container by 1:25 and mixed, as cleaning
Liquid is spare.
3) it detects antibody working solution: being prepared using in preceding 30min, measured as needed, by biotin labelled antibodies and experiment
Buffer is added in clean disposable container and is mixed by 1:1 000；When secondary experiment is finished.
4) marker working solution: preparing using in preceding 30min, measures as needed, and marker and test buffer are pressed 1:1
500 add in clean disposable container and mix；When secondary experiment is finished.
2. detecting step
1) 50 μ L calibration objects are pipetted with pipettor and sample sequentially adds in the reacting hole of micro reaction plate.Every hole is added again
50 μ L detect antibody marker working solution.
2) at 30 DEG C, oscillation incubation 90min.
3) it after being incubated for, is washed 6 times with cleaning liquid.
4) 100 μ L of enhancement solution is sequentially added into each reacting hole.
5) solid phase carrier detects after vibrating 5min at room temperature, completes to detect and analyzed in 30min.
Embodiment 3
A kind of component of time-resolved fluoroimmunoassay detection placenta growth factor reagent includes:
1) micro reaction plate: being coated with Streptavidin, and peridium concentration is 5 μ g/mL；
2) calibration object: calibration object contains 6 A, B, C, D, E, F concentration points, and calibration object A is zero-dose, calibration object B, C, D,
E, F contains people's recombinant placenta growth factor of series of concentrations gradient.
3) it captures antibody: capturing antibody containing biotin labeling placenta growth factor.
4) it detects antibody marker: detecting antibody containing europium label placenta growth factor.
5) test buffer: contain 0.02% in the tris-HCI buffer of 50mmol/L, pH 7.8
(v/v)300, the SignalBoost of the thimerosal of 20ppm, 10% (v/v)TMImmune signal reinforcing agent, 0.1%
(m/v) disodium ethylene diamine tetraacetate of the calf serum of PEG6 000,10% (v/v), 0.001% (m/v).
6) it is concentrated washing lotion: the Tween-20 of sodium chloride, 0.2mL/L containing 1.125g/L, 30ppm300 is anti-
The tris-HCI buffer of rotten agent.
7) enhancement solution: logical, glacial acetic acid, chelating agent, purified water containing Qula.
1. reagent prepares
1) micro reaction plate: reagent and required amount of micro reaction plate are balanced to room temperature (20~25 DEG C).It is remaining
Micro reaction plate be placed in time valve bag it is closed and in 2~8 DEG C save.
2) cleaning liquid: cleaning solution and purified water are added in clean container by 1:25 and mixed, as cleaning
Liquid is spare.
3) it captures antibody working solution: being prepared using in preceding 30min, measured as needed, by biotin labelled antibodies and experiment
Buffer is added in clean disposable container and is mixed by proper proportion, and 5 μ g/mL of antibody concentration is made；When secondary experiment is finished.
4) it detects antibody marker working solution: being prepared using in preceding 30min, measure as needed, will test antibody marker
It adds in clean disposable container and mixes by 1:1 200 with test buffer；When secondary experiment is finished.
2. detecting step
1) 100 μ L capture antibody working solution is pipetted with pipettor to sequentially add in the reacting hole of micro reaction plate.
2) micro reaction plate at room temperature, oscillation incubation 30min.
3) it after the first step is incubated for, is washed 4 times with cleaning liquid.
4) 100 μ L of calibration object, quality controling product and sample is sequentially added in the reacting hole of micro reaction plate.
5) at 30 DEG C, oscillation incubation 90min.
6) it after second step is incubated for, is washed 2 times with cleaning liquid.
7) detection 100 μ L of antibody marker working solution is sequentially added in the reacting hole of micro reaction plate.
8) at 30 DEG C, oscillation incubation 60min.
9) it after third step is incubated for, is washed 6 times with cleaning liquid.
10) 100 μ L of enhancement solution is sequentially added into each reacting hole.
11) solid phase carrier detects after vibrating 5min at room temperature, completes to detect and analyzed in 30min.
Embodiment 4
The reagent component of embodiment 4 and preparation are identical with embodiment 3, and detecting step is as follows:
1) 50 μ L capture antibody working solution is pipetted with pipettor to sequentially add in the reacting hole of micro reaction plate.
2) micro reaction plate at room temperature, oscillation incubation 15min.
3) it is respectively 50 μ L that calibration object, quality controling product and sample are sequentially added in the reacting hole of micro reaction plate.
4) at 30 DEG C, oscillation incubation 90min.
5) it after the first step is incubated for, is washed 4 times with cleaning liquid.
6) detection 100 μ L of antibody marker working solution is sequentially added in the reacting hole of micro reaction plate.
7) at 30 DEG C, oscillation incubation 60min.
8) it after second step is incubated for, is washed 6 times with cleaning liquid.
9) 100 μ L of enhancement solution is sequentially added into each reacting hole.
11) solid phase carrier detects after vibrating 5min at room temperature, completes to detect and analyzed in 30min.
Embodiment 5
The reagent component of embodiment 5 and preparation are identical with embodiment 3, and detecting step is as follows:
1) 100 μ L capture antibody working solution is pipetted with pipettor to sequentially add in the reacting hole of micro reaction plate.
2) micro reaction plate at room temperature, oscillation incubation 30min.
3) it after the first step is incubated for, is washed 4 times with cleaning liquid.
4) 50 μ L of calibration object, quality controling product and sample is sequentially added in the reacting hole of micro reaction plate, is added simultaneously
Detect 50 μ L of antibody marker working solution.
5) at 30 DEG C, oscillation incubation 90min.
6) it after second step is incubated for, is washed 6 times with cleaning liquid.
7) 100 μ L of enhancement solution is sequentially added into each reacting hole.
8) solid phase carrier detects after vibrating 5min at room temperature, completes to detect and analyzed in 30min.
Embodiment 6
The reagent component of embodiment 6 and preparation are identical with embodiment 3, and detecting step is as follows:
1) 50 μ L capture antibody working solution is pipetted with pipettor to sequentially add in the reacting hole of micro reaction plate.
2) micro reaction plate is at 30 DEG C, oscillation incubation 15min.
3) 50 μ L of calibration object, quality controling product and sample is sequentially added in the reacting hole of micro reaction plate.And every hole adds
Enter to detect 50 μ L of antibody marker working solution.
4) at 30 DEG C, oscillation incubation 90min.
5) it after being incubated for, is washed 4 times with cleaning liquid.
6) 100 μ L of enhancement solution is sequentially added into each reacting hole.
7) solid phase carrier detects after vibrating 5min at room temperature, completes to detect and analyzed in 30min.
Embodiment 7
A kind of component of time-resolved fluoroimmunoassay detection placenta growth factor reagent includes:
1) micro reaction plate: being coated with capture antibody, and peridium concentration is 5 μ g/mL；
2) calibration object: calibration object contains 6 A, B, C, D, E, F concentration points, and calibration object A is zero-dose, calibration object B, C, D,
E, F contains people's recombinant placenta growth factor of series of concentrations gradient.
3) it detects antibody: detecting antibody containing biotin labeling placenta growth factor.
4) it detects marker: containing europium labelled streptavidin.
5) test buffer: contain 0.02% in the tris-HCI buffer of 50mmol/L, pH 7.8
(v/v)300, the thimerosal of 20ppm, the SignalBoostTM immune signal reinforcing agent of 10% (v/v), 0.1%
(m/v) disodium ethylene diamine tetraacetate of the calf serum of PEG6 000,10% (v/v), 0.001% (m/v).
6) it is concentrated washing lotion: the Tween-20 of sodium chloride, 0.2mL/L containing 1.125g/L, 30ppm300 is anti-
The tris-HCI buffer of rotten agent.
7) enhancement solution: logical, glacial acetic acid, chelating agent, purified water containing Qula.
1. reagent prepares
1) micro reaction plate: reagent and required amount of micro reaction plate are balanced to room temperature (20~25 DEG C).It is remaining
Micro reaction plate be placed in time valve bag it is closed and in 2~8 DEG C save.
2) cleaning liquid: cleaning solution and purified water are added in clean container by 1:25 and mixed, as cleaning
Liquid is spare.
3) it detects antibody working solution: being prepared using in preceding 30min, measured as needed, by biotin labelled antibodies and experiment
Buffer is added in clean disposable container and is mixed by 1:1 000；When secondary experiment is finished.
4) marker working solution: preparing using in preceding 30min, measures as needed, will test marker and test buffer
It adds in clean disposable container and mixes by 1:1 500；When secondary experiment is finished.
2. detecting step
1) reacting hole that 100 μ L calibration objects, quality controling product and sample sequentially add micro reaction plate is pipetted with pipettor
In.
2) micro reaction plate at room temperature, oscillation incubation 90min.
3) it after the first step is incubated for, is washed 2 times with cleaning liquid.
4) detection 100 μ L of antibody working solution is sequentially added in the reacting hole of micro reaction plate.
5) at 30 DEG C, oscillation incubation 60min.
6) it after second step is incubated for, is washed 4 times with cleaning liquid.
7) 100 μ L of marker working solution is sequentially added in the reacting hole of micro reaction plate.
8) at 30 DEG C, oscillation incubation 15min.
9) it after third step is incubated for, is washed 6 times with cleaning liquid.
10) 100 μ L of enhancement solution is sequentially added into each reacting hole.
11) solid phase carrier detects after vibrating 5min at room temperature, completes to detect and analyzed in 30min.
Embodiment 8
The reagent component of embodiment 8 and preparation are identical with embodiment 7, and detecting step is as follows:
1) reacting hole that 100 μ L calibration objects, quality controling product and sample sequentially add micro reaction plate is pipetted with pipettor
In.
2) micro reaction plate at room temperature, oscillation incubation 90min.
3) it after the first step is incubated for, is washed 2 times with cleaning liquid.
4) detection antibody working solution and each 50 μ L of marker working solution are sequentially added in the reacting hole of micro reaction plate.
5) at 30 DEG C, oscillation incubation 60min.
6) it after second step is incubated for, is washed 6 times with cleaning liquid.
7) 100 μ L of enhancement solution is sequentially added into each reacting hole.
8) solid phase carrier detects after vibrating 5min at room temperature, completes to detect and analyzed in 30min.
Embodiment 9
The reagent component of embodiment 9 and preparation are identical with embodiment 7, and detecting step is as follows:
1) reacting hole that 50 μ L calibration objects, quality controling product and sample sequentially add micro reaction plate is pipetted with pipettor
In.Detection 50 μ L of antibody working solution is sequentially added in the reacting hole of micro reaction plate again.
2) micro reaction plate at room temperature, oscillation incubation 90min.
3) it after the first step is incubated for, is washed 4 times with cleaning liquid.
4) 100 μ L of marker working solution is sequentially added in the reacting hole of micro reaction plate.
5) at 30 DEG C, oscillation incubation 15min.
6) it after second step is incubated for, is washed 6 times with cleaning liquid.
7) 100 μ L of enhancement solution is sequentially added into each reacting hole.
8) solid phase carrier detects after vibrating 5min at room temperature, completes to detect and analyzed in 30min.
Embodiment 10
The reagent component of embodiment 10 and preparation are identical with embodiment 7, and detecting step is as follows:
1) 50 μ L samples are pipetted with pipettor to sequentially add in the reacting hole of micro reaction plate.50 μ L detection is added in every hole again
Antibody working solution and 50 μ L marker working solutions.
2) at 30 DEG C, oscillation incubation 90min.
3) it after being incubated for, is washed 6 times with cleaning liquid.
4) 100 μ L of enhancement solution is sequentially added into each reacting hole.
5) solid phase carrier detects after vibrating 5min at room temperature, completes to detect and analyzed in 30min.
One, the screening of antibody pair
Test method: cross match reaction is carried out to 6 kinds of antibody in such as table 1.The reactivity of each pair of antibody pair is being examined respectively
It surveys in the normal sample of calibration object A~F, the normal sample of pregnant early screening (early sieve) and second trimester screening (middle sieve) and is surveyed
Examination.
1 Subject antibodies information of table
Using reactive result of the different antibody conjugates in different samples such as table 2.
The different antibody of table 2 is to the reactivity in different samples
| Sample | a+b | a+c | a+e | d+e | b+d | d+c | f+c | f+b | f+e |
| A | 828 | 936 | 844 | 1487 | 604 | 604 | 3671 | 2487 | 2209 |
| B | 1983 | 2223 | 825 | 1543 | 641 | 743 | 8839 | 7543 | 7311 |
| C | 5600 | 5653 | 1008 | 1585 | 778 | 692 | 14428 | 13851 | 13880 |
| D | 21863 | 23203 | 1684 | 1782 | 1608 | 1442 | 25843 | 24821 | 21748 |
| E | 103432 | 111021 | 6178 | 2890 | 9422 | 6367 | 112887 | 12890 | 13484 |
| F | 745843 | 811971 | 63307 | 11592 | 44897 | 31611 | 437723 | 41592 | 43679 |
| Early sieve sample 1 | 6435 | 6923 | 5403 | 1622 | 1289 | 10997 | 8299 | 9132 | 6107 |
| Early sieve sample 2 | 5211 | 9445 | 4755 | 1871 | 1659 | 8447 | 8190 | 7123 | 4376 |
| Early sieve sample 3 | 5727 | 7087 | 4507 | 2052 | 12635 | 11891 | 5647 | 7993 | 4979 |
| Middle sieve sample 1 | 11788 | 13830 | 7853 | 2315 | 30118 | 8998 | 18928 | 14343 | 7797 |
| Middle sieve sample 2 | 13626 | 16485 | 9422 | 3247 | 35189 | 18230 | 18629 | 13522 | 7676 |
| Middle sieve sample 3 | 16917 | 20694 | 7965 | 2125 | 37099 | 3345 | 8747 | 7792 | 8897 |
It can be obtained from result, with mouse monoclonal antibody 655565 and the pairing of goat polyclonal antibodies 655170, mouse Dan Ke
Grand antibody 655565 is matched with goat polyclonal antibodies 446, and testing calibration product gradient is good, is had between middle sieve sample and early sieve sample
There is visible trend.Mouse monoclonal antibody 655565 and the pairing of rabbit monoclonal antibodies 625393 and rabbit monoclonal antibodies
625392 is poor with the pairing testing calibration product reactivity of rabbit monoclonal antibodies 625393, not strong with the affinity of sample.Rabbit monoclonal
The reactivity that antibody 625392 and goat polyclonal antibodies 655170 match testing calibration product A~F is weak, detects the early certain samples of sieve
Affinity is weak, and sensitivity is not high.Rabbit monoclonal antibodies 625392 and goat polyclonal antibodies 446 match testing calibration product A~F's
Reactivity is weak, and the resolution ratio for detecting early sieve sample and middle sieve sample is not high.Mouse monoclonal antibody 3931 and Goat polyclonal are anti-
The pairing of body 446 and mouse monoclonal antibody 3931 and goat polyclonal antibodies 655170 match the background values of testing calibration product A
Excessively high, calibration object is fitted concentration of specimens compared with mouse monoclonal antibody 655565 and the pairing of goat polyclonal antibodies 655170, mouse
Monoclonal antibody 655565 matches low, and school test sample this irregularities with goat polyclonal antibodies 446.Mouse monoclonal antibody
3931 is excessively high with the background values of the pairing of rabbit monoclonal antibodies 625393 testing calibration product A, and sample is sieved in detection and early sieves sample
Affinity is low, and resolution ratio is not high.Therefore selection mouse monoclonal antibody 655565 matches with goat polyclonal antibodies 655170, is small
Mouse monoclonal antibody 655565 and goat polyclonal antibodies 446 match, and continue optimization experiment.
Two, the performance test of kit reaction pattern.
It is capture antibody with mouse monoclonal antibody 655565, is that detection is anti-with goat polyclonal antibodies 446 or 655170
Body carries out following items test to Examples 1 to 10, can reach following performance indicator:
1. precision: the EP5-A2 file according to CLSI determines:
1) calibration object precision: kit calibration object (except zero-dose) B point, C point the coefficient of variation (CV) be no more than
20%；D point, E point, F point the coefficient of variation (CV) be no more than 15%.
2) withinrun precision: the quality-control product of detection high level, 3 intermediate value, low value concentration, variation within batch coefficient (CV) are no more than
10% (n=10).
3) betweenrun precision: the quality-control product with the reagent detection of three lot numbers with a median concentration, interassay coefficient of variation
(CV) it is no more than 15% (n=30).
2. detection lower bound (LLD): using zero-dose calibration object as sample, replication 20 times, calculate it and detect fluorescent value
Mean valueWith standard deviation (S), calculated using dose-response curveCorresponding concentration value is detection lower bound,
Detection lower bound should be better than 0.5pg/mL.
3. accuracy: within the scope of kit calibration object B~F, detecting enterprise with the mating calibration object of kit after calibration
Reference material, the measured value of reference material and the relative deviation of theoretical value should be in ± 10.0% ranges.
4. linear: the EP6-A file according to CLSI determines: within the scope of 5.0~10 500pg/mL, linearly dependent coefficient r
Not less than 0.990 0.
5. specificity: the EP7-A2 file according to CLSI determines.
Detect 1 0000pg/mL PlGF-1 (glycosylation), 1 000pg/mL PlGF-2 (glycosylation), 1 000pg/mL
PlGF-3 (being not glycosylated), 11 000pg/mL PlGF/VEGF heterodimers (being not glycosylated), 100 000pg/mL
VEGF165 (glycosylation) measures apparent cross reaction (%) and is respectively no higher than 45%, 30%, 25%, 0.1%, 0.1%.
6.HOOK effect: the PlGF sample of 150 000pg/mL of detection, detection fluorescent value are still higher than the highest of calibration object
The detection fluorescent value of value.
7. blank limits, (LoB), detection limit (LoD), quantitatively the EP17-A file of (LoQ): CLSI is determined.
It is 0.5pg/mL that blank, which limits (LoB),.Detection limit (LoD) is 2.0pg/mL.Quantitative (LoQ) is limited to 5.0pg/mL.When
Detected value is not detected in 0.5~2.0pg/mL, being construed to analyte；It, can when detected value is in 2.0~5.0pg/mL
To be construed to analyte, but cannot quantify；When detected value is in >=5.0pg/mL, there is analyte, can quantify, if desired can report
Uncertainty or quality objective.
8. interference is tested: haemolysis (hemoglobin≤180g/L), chylemia (triglycerides≤21.54mmol/L), Huang
Subcutaneous ulcer blood (bilirubin≤818 μm ol/L), 1 500 μm of ol/L paracetamol, 200 μm of ol/L ascoltins,
4.00mmol/L aspirin, 100nmol/L biotin, 310 μm of ol/L caffeines, 5.0mmol/L calcium, 5% (v/v) ethyl alcohol
(99.5%), 1 500nmol/L folic acid, 25 μm of ol/L gentamicins, 60g/L human serum albumins, 2 500 μm of ol/L isobutylbenzenes
Propionic acid, pH 6.8~9.3,45g/L total protein (gamma globulin), 6.5 μm of ol/L nicotines and 800IU/mL rheumatoid factor,
To the noiseless effect of this kit under concentrations above/degree.
3 kit all-round property testing of table
Wherein, table 3 be with goat polyclonal antibodies 446 be detect antibody result.By 3 test result comprehensive performance ratio of table
Compared with can obtain, embodiment 1 is opposite to have the higher range of linearity, and sensitivity, anti-interference are also stronger, preferred embodiment 1 it is dual anti-
The sandwich two step method of body optimizes.By embodiment 1 reaction pattern to calibration object A~F (concentration is respectively 0,20,100,500,
25 00,10 500pg/mL) dosage test is carried out, and make response curve (using X- principal axis transformation: LOG；Y- principal axis transformation: LOG_B,
Fitting algorithm: SPLINE.), as a result such as Fig. 1.
Three, the selection of the micro reaction plate peridium concentration of the reaction pattern of double-antibody sandwich two step method.
On optimized conditioned basic, peridium concentration is optimized: capture antibody is dissolved in 0.1mol/L, pH
4.5 phosphate buffer, compound concentration is the antibody coating buffer of 2,3,4,5,6 and 7 μ g/mL respectively, is wrapped to microwell plate
Quilt.The fluorescent value that the detection coated micropore of various concentration antibody is reacted with calibration object.The fluorescent value of calibration object A is background, is indicated
For N, N≤2000 are preferred；The ratio of calibration object B, F and calibration object A are respectively PB/N、PF/N。PB/ N shows more greatly resolution ratio more
Good, sensitivity is higher；PF/ N shows that more greatly detection range is wider.The testing result of different peridium concentrations is shown in Table 4.
The different peridium concentration test results of table 4
It is obtained from result above, under identical coating and sealing condition, the capture antibody concentration background values N that is tested <
2000, it meets the requirements.But the fluorescent value of calibration object B~F rises with peridium concentration in rising and downward trend, dense being coated with
When degree is 5.0 μ g/mL, reach maximum.This is because with peridium concentration less than 5.0 μ g/mL when, adsorb on micro reaction plate
Antibody is reduced, and is reduced with antigen reactive site, and the detection fluorescent value of calibration object declines with peridium concentration and is gradually reduced；However
With the increasing of peridium concentration, the antibody adsorbed on micro reaction plate increases, but increasing to a certain amount of (5.0 μ g/mL) may
The antibody of overlapping is caused also to increase, steric effect increases, and the fastness in conjunction with antibody response weakens.Therefore, antibody is most suitable
Peridium concentration is 5.0 μ g/mL.
Four, the selection of the micro reaction plate coating volume of the reaction pattern of double-antibody sandwich two step method.
On optimized conditioned basic, coating volume is optimized: being added in each hole of micro reaction plate respectively
50,100,150,200 μ L antibody coating buffers, then coating incubation and closing are carried out by the preparation method of solid phase reaction plate.Detection is not
The fluorescent value that the coated micropore of same volume antibody is reacted with calibration object.Analysis bag surveys CPs and P/N to Linear Experiment product by volume
Influence, choose background it is good, the P of calibration object BBThe coating volume that/N result reaches 2.1 or more is most suitable coating volume.It is examined
Survey the results are shown in Table 5.
The test result of the different coating volumes of table 5
Reaction volume be not be liquid volume in micropore, but in solid-phase immunoassay with the antibody of solid-phase coating or anti-
The part of original contact, this partial volume on earth how many, be difficult to measure, but fewer than total liquid volume in reaction micropore
It is more.Solid phase antigen antibody response betides liquid-solid phase interface, is likely to be in the gravitation distance of level-one associative key, is less thanDetermined antigen or antibody will enter this combination interface in liquid phase, need by diffusion or mass transfer.It may participate in
The antibody of association reaction or the concentration of antigen depend on may participate in the reaction volume of combination.Specificity in solid-phase immunoassay is anti-
The effect degree and speed of antigen-antibody reaction not only follow " law of mass action ", are also influenced by " molecule diffusion ".In general, solid
In phase immunoassays, antigen reaches the time required for balance in conjunction with antibody, with volume shared by liquid and interface antibody or resists
The increase of volume ratio shared by original receptor and increase.The additional amount of board-like sample is generally 100~200 μ L, can be appropriate as needed
Reaction volume is increased, the increase of sample reacting dose facilitates the raising of testability.Therefore, meeting detection range and sensitive
Under the conditions of degree, cost of material is saved, the coating volume that this research is selected is 150 μ L.
Five, the selection of the injection volume of the reaction pattern of double-antibody sandwich two step method.
On optimized conditioned basic, the sample volume of addition is optimized: being respectively 50,75 by volume,
Calibration object A~F of 100,125,150 μ L is added separately to compare the actual measurement of different volumes sample in ready reaction micropore
CPs.Selection detection background N is good, the P of calibration object BB/ N result is greater than 2.1 or more, and the actual measurement CPs and P/N of calibration object B~F is tied
It is most suitable injection volume that fruit ascensional range, which starts the injection volume to tend to balance,.As a result such as table 6.
The result of the different injection volume tests of table 6
Can be obtained from test result, with the increasing of injection volume, actual measurement CPs and the P/N result of calibration object B~F also with
Increase；But when injection volume is greater than 100 μ L, the P/N result ascensional range of calibration object B~F starts to tend to slow down, and when sample-adding
When volume is up to 100 μ L, the P of calibration object BB/ N result reaches balance not shadow up to 2.1 or more, in order to react in a relatively short period of time
The sensibility of test is rung, this research preferably selects 100 μ L according to test result injection volume.
Six, the selection of the reaction temperature of the reaction pattern of double-antibody sandwich two step method.
The sample that the addition of Human plactnta growth factor sterling is configured to various concentration in plasma sample is detected, as a result
Such as table 7.It can be obtained from table 7, the testing result after 25 DEG C tends towards stability, and bias is smaller between testing result.
Influence of the 7 differential responses temperature of table to pattern detection result
Seven, the selection of the frequency of oscillation of the reaction pattern of double-antibody sandwich two step method.
The sample that the addition of Human plactnta growth factor sterling is configured to various concentration in plasma sample is detected, as a result
Such as table 8.
Influence of the different frequencies of oscillation of table 8 to pattern detection result
It can be obtained from result, the testing result after 950rpm tends towards stability, and bias is smaller between testing result.
Eight, the selection in the reaction time of the reaction pattern of double-antibody sandwich two step method.
On optimized conditioned basic, for the sensitivity and specificity of improvement method, in order to avoid as far as possible
HOOK effect selects double-antibody sandwich two-step method.Detection method should apply under the conditions of common lab, and reaction preferably selects room temperature
(20~25 DEG C) condition.When immune response changes incubation time (30,60,90 and 120min) and the step 2 incubation of step 1 respectively
Between (30,45,60 and 90min) dissociation-enhancing duration of oscillation (0,1,2,3,4,5,10,20 and 30min), detect each calibration object
CPs is averaged.Selection detection background is good, the P of calibration object BB/ N result is greater than 2.1 or more, the actual measurement CPs of calibration object B~F
And the reaction time of the close balance of P/N result ascensional range is as the optimal reaction time.
It is 90min step 2 reaction time 60min when the reaction time of step 1, the actual measurement CPs of testing calibration product A~F becomes
In " saturation ", showing that reaction tends to completely, see Table 9 for details for testing result~and 10.When dissociation-enhancing oscillation 5min, calibration object A~F
Actual measurement CPs tended to balance, show Eu3+It sufficiently disintegrates down from Europium label chelate, is formed with enhancement solution ingredient
Stable compound, testing result are detailed in Fig. 1.
The testing result of the different step 1 incubation times of table 9
The testing result of the different step 2 incubation times of table 10
Nine, clinical sample testing result
The sample standard deviation that this test uses is from pregnant morning, the remaining sample of mid-term Prenatal Screening detection.According to " antenatal sieve
Look into technical specification ", have collected 163 First Trimester pregnancy serum samples, 172 pregnant second trimester pregnancy serum samples, pregnant woman
Age, average age was 28 years old at 20~38 years old, and through pregnant week calculating, (pregnant woman's menstruation rule person pregnant week was calculated with last menstrual period first day
Rise, irregular menses person determines pregnant week with ultrasound), early stage pregnant week is 9~13 weeks+6d, mid-term pregnant week be 14~20 weeks+6d, pregnant woman
Weight is in 35.5~98.6kg, average weight 54.1kg.All samples of this test use directly with intravenous blood collection in vacuum
In drying tube, after condensing, centrifuging and taking serum, 2~8 DEG C of preservations.Sample requirement: without piarhemia, haemolysis.Testing result such as 11 He of table
Table 12.
The pregnant early stage blood serum sample PlGF measurement result of table 11
12 second trimester serum sample PlGF measurement result of table
The clinical data is the reference median for establishing PlGF in different pregnant week mother serum, is clinic in female serum
PlGF normally whether judgement provide reference frame.
In addition, by determining one group of clinical sample (the remaining pregnant female serum sample of clinical examination), wherein 5 detection knots
Fruit is significantly lower than corresponding PlGF median when pregnant female blood sampling pregnant week, and 2 are nourished Down syndrome to make a definite diagnosis, and 3 clinics are examined
Breaking, there are eclampsias, intrauterine growth restriction for pregnant woman.
Finally it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, rather than the present invention is protected
The limitation of range is protected, although explaining in detail referring to preferred embodiment to the present invention, those skilled in the art are answered
Work as understanding, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the reality of technical solution of the present invention
Matter and range.
Claims (10)
1. a kind of detection kit of Human plactnta growth factor, which is characterized in that including specificity capture antibody and specificity
Detect antibody；The specificity capture antibody and the specific detection antibody are selected from following antibody: goat polyclonal antibodies
655170, mouse monoclonal antibody 655565, goat polyclonal antibodies 446, and the specificity capture antibody and described special
Property detection antibody conjugates Species origin answer difference；The mouse monoclonal antibody 655565 uses amino acid sequence for Ala21-
The Bacillus coli expression recombined human PlGF of Arg149 (Genbank Shelf number is P49763), is immunized obtained by mouse；The goat is more
Clonal antibody 655170 and goat polyclonal antibodies 446 are to use amino acid sequence for Ala21-Arg149 (Genbank Shelf number
For the Bacillus coli expression recombined human PlGF of CAA38698), immune goat gained.
2. detection kit according to claim 1, which is characterized in that further include solid phase carrier, marker, calibration object,
Cleaning solution, enhancement solution and test buffer；It is coated with the specificity capture antibody on the solid phase carrier or strepto- is affine
Element；The marker is that rare earth element and specific detection antibody or Streptavidin object form specific detection antibody marker
Or marked by streptavidin object；The calibration object is that the calibrated product dilution of people's recombinant placenta growth factor is diluted by concentration gradient
It forms.
3. detection kit according to claim 2, which is characterized in that the rare earth element is europium.
4. detection kit according to claim 2, which is characterized in that the calibration object dilution is 50mmol/L,
The tris-HCI buffer of pH7.8, containing 5% bovine serum albumin(BSA), 20ppm thimerosal, and
0.01% Sodium azide.
5. detection kit according to claim 2, which is characterized in that the cleaning solution be 1.125g/L sodium chloride,
The polysorbas20 of 0.02% (v/v), 30ppmThe tris-HCI buffer of 300 preservatives forms
Mixture.
6. detection kit according to claim 2, which is characterized in that the enhancement solution logical, glacial acetic acid, chelating for Qula
The mixture that agent, purified water form.
7. detection kit according to claim 2, which is characterized in that the test buffer is by buffer, anti-corrosion
The mixed solution that agent, immune signal reinforcing agent, protein and chelating agent form: the buffer is 50mmol/L, pH's 7.8
Tris-HCI buffer；The preservative is to contain 0.02% (v/v's)300, the sulphur of 20ppm
Willow mercury；The chelating agent is the disodium ethylene diamine tetraacetate of 0.001% (v/v)；The immune signal reinforcing agent is 5%~20%
(v/v) SignalBoostTMOne of immune signal reinforcing agent, 0.05%~0.2% (m/v) Macrogol 6000 or two
Kind；The protein is γ-ox ball egg of the mice serum of 0.1% (v/v), the calf serum of 10% (v/v), 0.5% (m/v)
White and 5% (m/v) one or more of bovine serum albumin(BSA).
8. the application method of detection kit according to claim 1, which is characterized in that use solid phase biological element Avidin
Method or bridge-type biotin-labeled pentylamine method or sandwich method；
The solid phase biological element Avidin method, steps are as follows:
A) the specificity capture antibody response of biotin labeling is added in solid-phase coating Streptavidin,
B) it is directly added into sample to be reacted, the reaction of specific detection antibody marker is added after washing,
Or sample is added after washing and is reacted, the reaction of specific detection antibody marker is added after washing,
Or sample and the reaction of specific detection antibody marker are directly added simultaneously,
Or sample and the reaction of specific detection antibody marker is added after washing simultaneously,
C) enhancement solution is added after washing, tests and analyzes；
The bridge-type biotin-labeled pentylamine method, steps are as follows:
A) solid-phase coating specificity captures antibody, and sample is added and is reacted,
B) the specific detection antibody reaction of biotin labeling is added after washing, it is anti-that marked by streptavidin object is added after washing
It answers,
Or biotin labeling specific detection antibody, the reaction of marked by streptavidin object is added after washing simultaneously,
Or it is directly added into the reaction of biotin labeling specific detection antibody, the reaction of marked by streptavidin object is added after washing,
Or biotin labeling specific detection antibody, the reaction of marked by streptavidin object are directly added simultaneously；
C) enhancement solution is added after washing, tests and analyzes；
The sandwich method, steps are as follows:
A) solid-phase coating specificity captures antibody,
B) sample is added to be reacted, the reaction of specific detection antibody marker is added after washing,
Or sample and the reaction of specific detection antibody marker are added simultaneously,
C) enhancement solution is added after washing, tests and analyzes.
9. the application method of detection kit according to claim 8, which is characterized in that using in sandwich method
A kind of: solid-phase coating specificity captures antibody, and sample is added and is reacted, washs, it is anti-that specific detection antibody marker is added
It answers, enhancement solution is added after washing, test and analyze；Specifically, steps are as follows:
(1) specificity capture antibody solid-phase coating: is dissolved in coating buffer into the packet containing 3~7 μ g/mL capture antibody
By liquid, 100~200 μ L coating buffer volumes is taken to carry out solid phase carrier coating；
(2) it is loaded: 50~150 μ L of test specimens being taken to be separately added into each solid phase carrier (being coated with specificity capture antibody) reaction position
In；
(3) it is incubated for: the solid phase carrier equipped with test specimens being carried out incubation 30~120 minutes on shaker, frequency of oscillation 750
~1150rpm, incubation temperature are 20~30 DEG C；
(4) board-washing: test specimens are discarded, and wash each reaction position 1~6 respectively with cleaning solution working solution (26 times of cleaning solution dilution)
It is secondary；
(5) antibody incubation: the antibody marker working solution of 50~150 μ L is separately added into each reaction position, and (specific detection is anti-
Body marker and test buffer are formulated by 1:1200, ready-to-use), and be incubated for 30~120 minutes on shaker, it shakes
Swinging frequency is 750~1150rpm, and incubation temperature is 20~30 DEG C；
(6) board-washing: marker working solution is discarded, and washs each reactant 2~10 times with cleaning solution；
(7) 50~150 μ L enhancement solutions, and oscillating reactions 1~30 minute signal enhancing: is added into each reaction position respectively；
(8) fluorescence signal detection and analysis: detected using time resolution immunofluorescence assay instrument, and according to detection data into
Row interpretation of result.
10. the method that detection kit according to claim 9 carries out placenta growth factor detection, which is characterized in that step
Suddenly coating buffer volume described in (1) is 150 μ L, and capture antibody concentration described in the coating buffer is 5 μ g/mL, and the coating is slow
Fliud flushing is 20mmol/L, the phosphate buffer of pH4.5；The additional amount of test specimens is 100 μ L in step (2)；Step (3), (5)
Incubation temperature during incubation is 30 DEG C, incubation time 60min, frequency of oscillation 1150rpm；Board-washing number in step (4)
It is 2 times；Board-washing number in step (6) is 6 times；The additional amount of enhancement solution is 100 μ L in step (7), and the reaction time is 5 points
Clock.
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| CN201910332813.3A Pending CN110031634A (en) | 2019-04-23 | 2019-04-23 | A kind of detection kit and its application method of Human plactnta growth factor |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111965352A (en) * | 2020-06-28 | 2020-11-20 | 广州市丰华生物工程有限公司 | Kit and method for screening progressive muscular dystrophy of newborn |
| CN113834942A (en) * | 2021-11-02 | 2021-12-24 | 河北特温特生物科技发展有限公司 | Placenta growth factor quality control product and preparation method thereof |
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| CN103235142A (en) * | 2013-04-28 | 2013-08-07 | 成都中医药大学 | First pregnancy screening kit for pregnant woman |
| CN103869073A (en) * | 2014-04-01 | 2014-06-18 | 广州市丰华生物工程有限公司 | Double-labeling time-resolved fluorescence immunoassay method and kit for HIV (human immunodeficiency virus) antibody and HIV-1p24 antigen |
| CN104730247A (en) * | 2015-03-12 | 2015-06-24 | 广州市丰华生物工程有限公司 | Kit suitable for rapidly detecting AMH and INHB by using double-tagging time resolution fluorescence immunoassay method and use method of kit |
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| CN102224422A (en) * | 2008-11-20 | 2011-10-19 | 珀金埃尔默健康科学公司 | Method for determining the risk of preeclampsia using pigf-2 and pigf-3 markers |
| CN103235142A (en) * | 2013-04-28 | 2013-08-07 | 成都中医药大学 | First pregnancy screening kit for pregnant woman |
| CN103869073A (en) * | 2014-04-01 | 2014-06-18 | 广州市丰华生物工程有限公司 | Double-labeling time-resolved fluorescence immunoassay method and kit for HIV (human immunodeficiency virus) antibody and HIV-1p24 antigen |
| CN104730247A (en) * | 2015-03-12 | 2015-06-24 | 广州市丰华生物工程有限公司 | Kit suitable for rapidly detecting AMH and INHB by using double-tagging time resolution fluorescence immunoassay method and use method of kit |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111965352A (en) * | 2020-06-28 | 2020-11-20 | 广州市丰华生物工程有限公司 | Kit and method for screening progressive muscular dystrophy of newborn |
| CN113834942A (en) * | 2021-11-02 | 2021-12-24 | 河北特温特生物科技发展有限公司 | Placenta growth factor quality control product and preparation method thereof |
| CN113834942B (en) * | 2021-11-02 | 2024-02-02 | 河北特温特生物科技发展有限公司 | Placenta growth factor quality control product and preparation method thereof |
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