CN110514828A - A kind of immunochromatographydetection detection card and preparation method thereof of quick detection pregnant women placental growth factor - Google Patents
A kind of immunochromatographydetection detection card and preparation method thereof of quick detection pregnant women placental growth factor Download PDFInfo
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- CN110514828A CN110514828A CN201910918350.9A CN201910918350A CN110514828A CN 110514828 A CN110514828 A CN 110514828A CN 201910918350 A CN201910918350 A CN 201910918350A CN 110514828 A CN110514828 A CN 110514828A
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- detection
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- surface active
- fluorescent latex
- plgf
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- 238000001514 detection method Methods 0.000 title claims abstract description 156
- 102100035194 Placenta growth factor Human genes 0.000 title claims abstract description 148
- 101000595923 Homo sapiens Placenta growth factor Proteins 0.000 title claims abstract description 34
- 238000002360 preparation method Methods 0.000 title claims description 41
- 238000012360 testing method Methods 0.000 claims abstract description 57
- 241000283973 Oryctolagus cuniculus Species 0.000 claims abstract description 38
- 101150062285 PGF gene Proteins 0.000 claims abstract 9
- 239000004816 latex Substances 0.000 claims description 73
- 229920000126 latex Polymers 0.000 claims description 73
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 claims description 48
- 238000002372 labelling Methods 0.000 claims description 47
- 241000196324 Embryophyta Species 0.000 claims description 38
- 239000011248 coating agent Substances 0.000 claims description 30
- 238000000576 coating method Methods 0.000 claims description 30
- 238000003756 stirring Methods 0.000 claims description 29
- 239000007788 liquid Substances 0.000 claims description 21
- 229910021538 borax Inorganic materials 0.000 claims description 20
- 239000004328 sodium tetraborate Substances 0.000 claims description 20
- 239000007853 buffer solution Substances 0.000 claims description 19
- 150000001718 carbodiimides Chemical class 0.000 claims description 19
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 16
- 239000006228 supernatant Substances 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 238000002156 mixing Methods 0.000 claims description 13
- 238000006243 chemical reaction Methods 0.000 claims description 12
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- KZRXPHCVIMWWDS-AWEZNQCLSA-N (4S)-4-amino-5-dodecanoyloxy-5-oxopentanoic acid Chemical compound CCCCCCCCCCCC(=O)OC(=O)[C@@H](N)CCC(O)=O KZRXPHCVIMWWDS-AWEZNQCLSA-N 0.000 claims description 10
- 241001502050 Acis Species 0.000 claims description 10
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 10
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims description 10
- YRIUSKIDOIARQF-UHFFFAOYSA-N dodecyl benzenesulfonate Chemical compound CCCCCCCCCCCCOS(=O)(=O)C1=CC=CC=C1 YRIUSKIDOIARQF-UHFFFAOYSA-N 0.000 claims description 10
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- 238000010521 absorption reaction Methods 0.000 claims description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical class OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 6
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 6
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- 235000010339 sodium tetraborate Nutrition 0.000 claims description 2
- ATHHXGZTWNVVOU-UHFFFAOYSA-N N-methylformamide Chemical compound CNC=O ATHHXGZTWNVVOU-UHFFFAOYSA-N 0.000 claims 2
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims 1
- FFAXGWPUPHCFAA-UHFFFAOYSA-L [Na+].C(C=C)(=O)[O-].C(C=C)(=O)[O-].C(CN)N.[Na+] Chemical compound [Na+].C(C=C)(=O)[O-].C(C=C)(=O)[O-].C(CN)N.[Na+] FFAXGWPUPHCFAA-UHFFFAOYSA-L 0.000 claims 1
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- 108010082093 Placenta Growth Factor Proteins 0.000 description 105
- 238000011084 recovery Methods 0.000 description 16
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 15
- 239000011734 sodium Substances 0.000 description 10
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 9
- 229910052708 sodium Inorganic materials 0.000 description 9
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 8
- 235000019082 Osmanthus Nutrition 0.000 description 8
- 241000333181 Osmanthus Species 0.000 description 8
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- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 4
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- 210000002889 endothelial cell Anatomy 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 3
- 239000010452 phosphate Substances 0.000 description 3
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 102000016548 Vascular Endothelial Growth Factor Receptor-1 Human genes 0.000 description 2
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- 230000002482 anti-endothelial effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
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- 210000004204 blood vessel Anatomy 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
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- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
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- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- 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
-
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
Abstract
The present invention relates to a kind of immunochromatographydetection detection cards of quickly detection pregnant women placental growth factor, including test strips, including test strips, the test strips include detection line and nature controlling line, it is coated with the anti-human PLGF monoclonal antibody of one plant of mouse in the detection line, is coated with goat-anti rabbit polyclonal antibody on the nature controlling line.The immunochromatographydetection detection card of quick detection pregnant women placental growth factor provided by the present invention, can be used for the detection of serum, blood plasma and whole blood, for predicting the diagnosis of preeclampsia related disease.
Description
Technical field
The invention belongs to in-vitro diagnosis fields, are related to a kind of immunochromatography detection of quickly detection pregnant women placental growth factor
Card and preparation method thereof.
Background technique
PLGF (placenta growth factor) is a member in vascular endothelial growth factor (VEGF) family, and molecular structure is sugar
Albumen homodimer molecule.It is a kind of assignment of genes gene mapping in the glycoprotein of 14q24q31, by the α chain of 1 69kD and the β of 34kD
Chain connects to form dimer by disulfide bond.Its base sequence and VEGF have high homology.By the alternative splicing of mRNA,
PLGF can produce 4 kinds of different subtypes: PLGF-1, PLGF-2, PLGF-3 and PLGF-4.
The biological function of PLGF is activated by specific bond its receptor VEGFR-1.VEGFR-1 has very strong
Biological activity also influences being divided into for endothelial cell in conjunction with the effect that can mediate endothelial cell and stroma cell after its ligand
It is ripe.PLGF can promote trophoblastic proliferation and differentiation, inducible endothelial cell proliferation when early pregnancy to migrate, anti-endothelial cell apoptosis,
And can increase the permeability of blood vessel, the biological activity of the VEGF of low concentration can be enhanced, be to participate in kinds of tumors angiogenesis
Important angiogenic factors.
PLGF is a kind of marker of high degree of specificity, and PLGF level is aobvious when placenta syncytiotrophoblast has oxygen supply pressure
Writing reduces, and detection pregnant woman blood PlGF level can be used to assess placental insufficiency, and to preeclampsia caused by thus into
Row prediction, identification and Treatment monitoring.
Summary of the invention
In view of this, the main purpose of the present invention is to provide a kind of immune layers of quickly detection pregnant women placental growth factor
Analysis detection card and preparation method thereof.
To achieve the goals above, the present invention provides the following technical scheme that
A kind of immunochromatographydetection detection card of quick detection pregnant women placental growth factor, including test strips, the test strips packet
Detection line and nature controlling line are included, the anti-human PLGF monoclonal antibody of one plant of mouse is coated in the detection line, is coated on the nature controlling line
There is goat-anti rabbit polyclonal antibody.
In a concrete scheme of the invention, wherein the test strips further include PVC board, it is fixed in the PVC board
Sequentially connected sample pad, labeling pad, coating pad and water absorption pad, the packet, which is covered with, is successively arranged detection line and nature controlling line,
The sample pad and labeling pad are connected as one.
In a concrete scheme of the invention, wherein the coating pads and is connected with marker close to one end of detection line
Pad, one end close to nature controlling line are connected with water absorption pad.
In a concrete scheme of the invention, wherein being coated with the anti-human PLGF Dan Ke of another plant of mouse in the labeling pad
The fluorescent latex microballoon of the surface active of the fluorescent latex microballoon and rabbit igg label of the surface active of grand antibody label, it is described another
The surface active of the anti-human PLGF labeling of monoclonal antibody of one plant of mouse fluorescent latex microballoon and rabbit igg label surface active it is glimmering
The molar ratio of light latex beads is 1:(0.2~4).
In a concrete scheme of the invention, wherein the immunochromatography of the quick detection pregnant women placental growth factor is examined
Surveying card further includes that getting stuck for test strips is set for card.
In a concrete scheme of the invention, wherein described get stuck includes:
Kerve is connected to the PVC board；
Upper cover is connected to the kerve, the well for being loaded in the sample pad is provided on the upper lid；
Observation window is set on lid and for detection line and the acquisition of the data of nature controlling line.
A kind of preparation method of the immunochromatographydetection detection card of quick detection pregnant women placental growth factor, comprising the following steps:
1) preparation of coating pad: the anti-human PLGF monoclonal antibody of one plant of mouse and goat-anti rabbit polyclonal antibody are coated with arrive respectively
On nitrocellulose filter, drying for standby；
2) preparation of labeling pad: by the fluorescent latex of the surface active of the anti-human PLGF labeling of monoclonal antibody of another plant of mouse
After the fluorescent latex microballoon mixing of the surface active of microballoon and rabbit igg label, it is sprayed on glass fibre element film, drying for standby；
3) test strips are assembled: the bonding coating pad in PVC board, and overlapped in the one end for the nature controlling line being covered with close to the packet
Water absorption pad, overlap joint labeling pad and its sample pad of connection in the one end for the detection line being covered with close to the packet；Then it is cut
At the test strips of required width, the reagent strip is put into gets stuck later.
In a concrete scheme of the invention, wherein the fluorescent latex microballoon of the surface active passes through following steps system
:
1) take surfactant that boric acid-borax buffer solution that pH value is 8~10 is added (comprising 0.5wt%~3wt%'s
PEG2000 in), dimethylformamide, N, N '-dicyclohexylcarbodiimide and n-hydroxysuccinimide are added, stirring is anti-
It answers；
2) it takes surface to have the dispersion liquid of the fluorescent latex microballoon of carboxyl, (includes with boric acid-borax buffer solution
The PEG2000 of 0.5wt%~3wt%) adjust pH to 8~10 after, be added in the resulting product of step 1), stirred at 25 DEG C anti-
1~5h is answered, after completion of the reaction, centrifugation removal supernatant obtains the fluorescent latex microballoon of surface active, buffered with boric acid-borax
Solution redissolves spare.
In a concrete scheme of the invention, wherein the surfactant includes N, the bis- lauroyl second two of N'-
Amine diacrylate sodium, polyethyleneglycol moon esters of silicon acis, dodecyl benzene sulfonate and lauroyl glutamate, four weight ratio
For (0.5~6): (4~9): (0.8~3): (5~14)；The fluorescent latex microballoon of the surfactant and the amino surface
Weight ratio be (0.5~120): 1.
In a concrete scheme of the invention, wherein the surface of the anti-human PLGF labeling of monoclonal antibody of another plant of mouse
The fluorescent latex microballoon of activation is made by following steps:
The fluorescent latex microballoon of surface active is taken to be added in carbodiimides (EDC) and n-hydroxysuccinimide (NHS)
2~6h is stirred at room temperature, the anti-human PLGF monoclonal antibody of another plant of mouse is then added, stirs 1~4h at room temperature, add 10~
50mg BSA confining liquid, continues 1~4h of stirring；At 2~8 DEG C, it is centrifuged 30min according to the revolving speed of 11000r/min, in removing
Clear liquid；Finally, being redissolved solid sediment with the phosphate buffer solution (pH=7.4) of 0.01M~0.5M, add
Proclin300 is saved for use at 4 DEG C.
In a concrete scheme of the invention, wherein the fluorescent latex microballoon of the surface active and another strain of mouse are anti-human
The mass ratio of PLGF monoclonal antibody is 1:(0.01~4).
Beneficial effects of the present invention:
1, the immunochromatographydetection detection card of quick detection pregnant women placental growth factor provided by the present invention, can be used for blood
Clearly, the detection of blood plasma, whole blood, for predicting the diagnosis of preeclampsia related disease；
2, the immunochromatographydetection detection card of quick detection pregnant women placental growth factor provided by the present invention, sample dosage
Small, detection can be completed within 5~20min, and linear detection range is 10pg/mL~5000pg/mL, greatly improve inspection
Survey efficiency.
3, the immunochromatographydetection detection card of quick detection pregnant women placental growth factor provided by the present invention, high sensitivity,
Stability is strong, the range of linearity is wide, has excellent accuracy and precision.
4, the immunochromatographydetection detection card of quick detection pregnant women placental growth factor provided by the present invention, in sample collection
Afterwards, without falling into a long wait, pattern detection can be carried out, easy to operate, the sample process time is short, reduces time cost, fits
It is detected for clinical quick diagnosis.
5, the immunochromatographydetection detection card of quick detection pregnant women placental growth factor provided by the present invention, by using table
The fluorescent latex microballoon of face activation overcomes lacking for fluorescent dye poor sensitivity on the market and wet type fluorescent microsphere stability difference
It falls into, can guarantee that intergranular relative distance is not easy to reunite, be not necessarily to buffer, can be redissolved at once after sample to be tested is added and smooth
Chromatography.
Detailed description of the invention
Fig. 1 is the immuno-chromatographic test paper strip schematic diagram of quick detection pregnant women placental growth factor provided by the present invention；
Fig. 2A is in one kind in the immunochromatographydetection detection card of quick detection pregnant women placental growth factor provided by the present invention
The schematic diagram of internal structure of lid；
Fig. 2 B is a kind of bottom in the immunochromatographydetection detection card of quick detection pregnant women placental growth factor provided by the present invention
The schematic diagram of internal structure of slot；
Fig. 3 is the calibration curve of pregnant women placental growth factor in the embodiment of the present invention 1；
Fig. 4 is the calibration curve of pregnant women placental growth factor in the embodiment of the present invention 2；
Fig. 5 is the calibration curve of pregnant women placental growth factor in the embodiment of the present invention 3；
Fig. 6 is the calibration curve of pregnant women placental growth factor in the embodiment of the present invention 4；
Wherein, 1-PVC plate, 2- coating pad, 3- labeling pad, 4- water absorption pad, 5- detection line, 6- nature controlling line, 7- marker
Junction, 8- sample, 9- sample pad, 11- upper cover, 12- kerve, 13- well, 14- observation window, 15- test strips placement region,
16- positioning column, 17- location hole, the first limited section 18-, the second limited section 19-, 20- third limited section.
Specific embodiment
For a further understanding of the present invention, the preferred embodiment of the invention is described below with reference to embodiment, still
It should be appreciated that these descriptions are only further explanation the features and advantages of the present invention, rather than to the claims in the present invention
Limitation.
It is purchase if not following material or reagent illustrate.
Embodiment 1:
The preparation of 1.PLGF immunochromatographydetection detection card
1) preparation of the fluorescent latex microballoon of surface active:
The bimonthly osmanthus Ethylene Diamine diacrylate sodium of N, N'-, polyethyleneglycol moon esters of silicon acis, dodecyl benzene sulfonate and
Lauroyl glutamate, four weight ratio are 0.5:4:0.8:5.
1. taking surfactant (the poly- second two of the bimonthly osmanthus Ethylene Diamine diacrylate sodium of the N comprising 0.5mg, N'-, 4mg
Single month esters of silicon acis of alcohol, the dodecyl benzene sulfonate of 0.8mg and the lauroyl glutamate of 5mg) 0.2mol/L, pH=is added
In 8.4 alkali borate buffer solution (PEG2000 comprising 0.5wt%~3wt%), add 1mL dimethylformamide,
The N of 1mg, N '-dicyclohexylcarbodiimide and 0.5mgN- HOSu NHS, carry out under the mixing speed of 120r/min
Reaction；
2. taking 1mL, fluorescent latex microballoon dispersion liquid of the surface containing 1wt% with carboxyl (is purchased from Shanghai Zhen Zhun biotechnology
Co., Ltd), (include 0.5wt%~3wt%'s with boric acid-borax buffer solution of 10mL, 0.2mol/L, pH=8.4
PEG2000 after) dilution is uniformly dispersed, step is added to 1. in resulting product, is stirred under 25 DEG C, the mixing speed of 120r/min
Reaction 5h is mixed, after completion of the reaction, according to the revolving speed centrifugation removal supernatant of 12000r/min, obtains the fluorescent latex of surface active
Microballoon, with 0.2mol/L, boric acid-borax buffer solution of pH=8.4 is redissolved spare to 1mL.
2) the fluorescent latex microballoon of the anti-human PLGF labeling of monoclonal antibody surface active of another plant of mouse
Take the fluorescent latex microballoon of the surface active prepared in 0.5mL step 1) that 1mg carbodiimides (EDC) is added, 1mg
N-hydroxysuccinimide (NHS), stirs 3h under room temperature, the revolving speed of 120r/min, and it is anti-human that another plant of mouse of 200 μ L is then added
PLGF monoclonal antibody stirs 2h under room temperature, the revolving speed of 120r/min, adds 20mg BSA confining liquid, continues to stir 2h.
At 2~8 DEG C, it is centrifuged 30min according to the revolving speed of 11000r/min, removes supernatant.Finally, with the phosphate buffer solution of 0.2M
(pH=7.4) solid sediment is redissolved to 1mL, the Proclin300 for adding 1 μ L is saved for use at 4 DEG C.
3) the fluorescent latex microballoon of rabbit igg polyclonal antibody label surface active
Take the fluorescent latex microballoon of the surface active prepared in 0.5mL step 1) that 1mg carbodiimides (EDC) is added, 1mg
N-hydroxysuccinimide (NHS), stirs 3h under room temperature, the revolving speed of 120r/min, and it is more that 200 μ L mg sheep rabbit iggs are then added
Clonal antibody stirs 2h under room temperature, the revolving speed of 120r/min, adds 20mg BSA confining liquid, continues to stir 2h.2~8
At DEG C, it is centrifuged 30min according to the revolving speed of 11000r/min, removes supernatant.Finally, with the phosphate buffer solution (pH=of 0.2M
7.4) solid sediment is redissolved to 1mL, the Proclin300 for adding 1 μ L is saved for use at 4 DEG C.
4) preparation of coating pad
The anti-human PLGF monoclonal antibody of one plant of mouse and goat-anti rabbit polyclonal antibody are used to the phosphate buffer of 0.2M respectively
(pH=7.4) it is diluted to 1mg/mL, carries out drawing film on nitrocellulose filter (NC film) with film gold spraying instrument is drawn.It is anti-containing one plant of mouse
The conduct detection line (T line) 5 of people's PLGF monoclonal antibody is used as nature controlling line (C line) 6 containing goat-anti rabbit polyclonal antibody, so
Afterwards at 37 DEG C, coating pad is made in dry 4h in the environment of humidity < 30%.
5) preparation of labeling pad
1. the phosphate buffer (pH=7.4) by glass fibre element film in 0.2M impregnates 46h, then dry at 35 DEG C
6h, it is spare.
2. the fluorescent latex of the surface active for the anti-human PLGF labeling of monoclonal antibody of another plant of mouse that molar ratio is 1:1 is micro-
Ball and rabbit igg label surface active fluorescent latex microballoon after mixing, according to the velocity spray of 5 μ L/cm in glass fibers
It ties up on plain film, is then placed at 37 DEG C dry 2h and labeling pad 3 is made.Another plant of anti-human PLGF of mouse is coated in labeling pad
The ground of the fluorescent latex microballoon of the surface active of fluorescent latex microballoon and the rabbit igg label of the surface active of labeling of monoclonal antibody
Side is called marker junction 7.
The purpose of setting flag object junction 7 is: blood sample 8 to be measured being added drop-wise in sample pad 9, in water absorption pad 4
Under the action of, blood sample chromatographs forward, and the PLGF in the blood sample of marker junction 7 and marker junction are coated with
The anti-human PLGF labeling of monoclonal antibody of another plant of mouse surface active the reaction of fluorescent latex microballoon, substance after reaction, not
Fluorescent latex microballoon, the fluorescent latex of rabbit igg label for participating in the anti-human PLGF labeling of monoclonal antibody of another plant of mouse of reaction are micro-
Ball continues to chromatograph with blood sample, and the table of the anti-human PLGF labeling of monoclonal antibody of another plant of mouse of reaction is participated at detection line
Face activation fluorescent latex microballoon and the coated anti-human PLGF monoclonal antibody reactive of one plant of mouse of detection line and rest on detection line,
The fluorescent latex microballoon that rabbit igg marks at nature controlling line reacts with the coated goat-anti rabbit polyclonal antibody of nature controlling line and rests on matter
Line is controlled, other substances continue to chromatograph, finally by the signal (note of the fluorescent microsphere of fluorescence immune chromatography instrument difference acquisition testing line
Make T) and nature controlling line fluorescent microsphere signal (being denoted as C), calculate T/C value, read blood sample from the standard curve of PLGF
The concentration of middle PLGF.
6) assembling of immunochromatographydetection detection card
Then the bonding coating pad 2 first in PVC board 1 overlaps water suction in one end of the nature controlling line 6 on coating pad 2
Pad is cut into 4mm with cutting machine in one end overlap joint labeling pad 3 of the detection line 5 close to coating pad 2 and its sample pad 9 of connection
Test strips are put into getting stuck and are prepared into the detection of pregnant women placental growth factor immunochromatography by the test strips (see Fig. 1) of ± 0.1mm
Card.
It is described to get stuck selected from the prior art, for example, described get stuck (as shown in Fig. 2A, Fig. 2 B) may include: kerve 12,
It is connected to the PVC board 1；Upper cover 11 is connected to the kerve 12, is provided in the upper cover 11 for the sample pad
The well 13 being loaded on 9；Observation window 14 is set in upper cover 11, is acquired for detection line 5 and the data of nature controlling line 6.
As shown in Figure 2 B, the kerve 12 includes: symmetrical multiple location holes 17, Duo Gesuo positioned at its inner surface
It states between location hole 17 equipped with multiple for limiting the first limited section 18 of test strips transverse shifting and for limiting test strips
Second limited section 19 of longitudinal movement；Symmetrically arranged first limited section 18 is enclosed with second limited section 19 is set as paper slip
Placement region 15 (dashed region), for placing test strips；
As shown in Figure 2 A, the upper cover 11 includes: the multiple positioning columns 16 matched with multiple location holes 17, in this way
With the use of upper cover 11 and kerve 12 to be fixed together；The upper cover 11 further includes for limiting moving up and down for test strips
Third limited section 20.
The top of the coating pad 2 is equipped with the observation window 14 for data acquisition, to expose all detection line 5 and matter
Line 6 is controlled, for collecting its testing result；And the observation window 14 is opened in the upper cover 11 and test strips placement region 15
The corresponding position in middle part.The upper cover 11 offers well at position corresponding with the sample pad 9, to be used for sample
Sample 8 is added dropwise on product pad 9.The detection line is apart from well 15-25mm.
2. detection
The sample to be tested of 60 μ L is taken to be added drop-wise to the well 13 of the PLGF immunochromatographydetection detection card of step 1 preparation (to examination
The sample pad 9 of paper slip) in sample to be tested, be stored at room temperature 15min, PLGF immunochromatographydetection detection card be then inserted into fluorescence immunoassay
Analyzer is detected, and can get testing result immediately.
3. the evaluation of test strips
1) the PLGF calibration object (0,10,40,200,1000,5000pg/mL) for configuring various concentration, with 1 step of the present embodiment
The PLGF immunochromatographydetection detection card of rapid 1 preparation is successively measured according to the detection process of step 2, and blank detection line is not higher than
10pg/mL, detection range are 10~5000pg/mL.Testing result is as shown in table 1, using concentration of specimens as abscissa, detection line with
The ratio (T/C) of nature controlling line is ordinate, is carried out curve fitting with logistic (four parameters), degree of fitting R2Respectively
0.9995, n=6, the PLGF calibration curve of preparation as shown in figure 3, T/C value and calibration object in the model that concentration is 10-5000pg/mL
It is had good correlation in enclosing.
1 detection data of table
| Normal concentration pg/mL | 0 | 10 | 40 | 200 | 1000 | 5000 |
| T/C value | 0.1744 | 0.6534 | 0.9755 | 1.3546 | 1.8535 | 2.2356 |
2) quantitative limit
Quantitative limit (limit of quantitation, LoQ), referring to the EP17-A2 file of CLSI publication.
According to the allowable error of clinical requirement and External quality evaluation, set the overall error target of this reagent PLGF as
30%.In the case where not considering analysis deviation, allow overall error (TEa)=2CV, i.e. CV=1/2TEa.PLGF concentration exists
The CV detected when 10.00pg/mL is 12.97%, less than 15%, meets quality objective requirement, therefore LoQ=10.00pg/mL,
Show the sensitivity with higher of PLGF fluorescent microsphere immunochromatographydetection detection card.
3) precision
The PLGF test strip of three batches is extracted, detectable concentration is the sample progress of 40,200,1000pg/mL respectively
Detection, each concentration point are measured in parallel 10 times, calculate the variation within batch coefficient and batch variation system of three lot number test strips
Number, between criticizing as can be seen from Table 2 and variation within batch coefficient is respectively less than 13.53%, illustrates PLGF fluorescence immune chromatography detection card
Precision it is higher.
2 detection data of table
4) rate of recovery
Taking three batch concentration is the PLGF calibration object of 5000pg/mL, is added separately to concentration close to 0 negative sample
In, wherein the volume ratio of the PLGF calibration object being added and negative sample is 1:9, replication 3 times, calculate the rate of recovery.Three batches
The result of the sample calibration product rate of recovery is respectively 97.45%, 100.99%, 97.39%, the rate of recovery 90%-110% it
Between, illustrate that measurement result complies with standard.
5) control experiment
20 clinical samples are collected, are detected using PLGF immunochromatographydetection detection card manufactured in the present embodiment and the PLGF of Roche
Experiment is compared in kit, and experimental result is shown in Table 3.
3 detection data of table
As can be seen that PLGF immunochromatographydetection detection card prepared by the embodiment of the present invention 1 is rechecked and surveyed from the testing result of table 3
Three times, result of the detection 1 respectively with detection 2, detection 3 carries out correlation analysis, and regression equation is respectively as follows: y=0.98479x-
3.06498 R2=0.9998；Y=1.0101x-3.1388, R2=0.9996；Show PLGF immunochromatography prepared by embodiment 1
Detection card has good stability.PLGF immunochromatographydetection detection card and Roche human placental growth factor detection kit prepared by embodiment 1
Result carry out correlation analysis, regression equation are as follows: y=0.9834x+22.577, R2=0.9923, n=20 show the present invention
PLGF immunochromatographydetection detection card and the testing result of Roche PLGF detection kit prepared by embodiment 1 is significant related.Therefore originally
The PLGF immunochromatographydetection detection card of invention can satisfy the demand of adjuvant clinical diagnosis.
Embodiment 2
The preparation of 1.PLGF immunochromatographydetection detection card
1) preparation of the fluorescent latex microballoon of surface active:
The bimonthly osmanthus Ethylene Diamine diacrylate sodium of N, N'-, polyethyleneglycol moon esters of silicon acis, dodecyl benzene sulfonate and
Lauroyl glutamate, four weight ratio are 5:7:2:8.
1. taking the surfactant (polyethylene glycol of the bimonthly osmanthus Ethylene Diamine diacrylate sodium of the N comprising 5mg, N'-, 7mg
Single month esters of silicon acis, the dodecyl benzene sulfonate of 2mg and the lauroyl glutamate of 8mg) 0.2mol/L is added, pH=8.8's
In alkali borate buffer solution (PEG2000 comprising 0.5wt%~3wt%), 1mL dimethylformamide, 1mg are added
N, N '-dicyclohexylcarbodiimide and 0.5mgN- HOSu NHS, are reacted under the mixing speed of 120r/min；
2. taking 1mL, contain 1wt%, fluorescent latex microballoon dispersion liquid of the surface with carboxyl (is purchased from Shanghai Zhen Zhun biotechnology
Co., Ltd), (include 0.5wt%~3wt%'s with boric acid-borax buffer solution of 10mL, 0.2mol/L, pH=8.4
PEG2000 after) dilution is uniformly dispersed, step is added to 1. in resulting product, it is anti-under 25 DEG C, the mixing speed of 120r/min
5h is answered, after completion of the reaction, supernatant is removed according to the revolving speed centrifugation 30min of 12000r/min, obtains the fluorescence cream of surface active
Glue microballoon, with 0.2mol/L, boric acid-borax buffer solution of pH=8.4 is redissolved spare to 1mL.
2) the fluorescent latex microballoon of the anti-human PLGF labeling of monoclonal antibody surface active of another plant of mouse
Take the fluorescent latex microballoon of the surface active prepared in 0.5mL step 1) that 1mg carbodiimides (EDC) is added, 1mg
N-hydroxysuccinimide (NHS), stirs 4h under room temperature, the revolving speed of 120r/min, and it is anti-human that another plant of mouse of 500 μ L is then added
PLGF monoclonal antibody stirs 4h under room temperature, the revolving speed of 120r/min, adds 50mg BSA confining liquid, continues to stir 4h.
At 2~8 DEG C, it is centrifuged 30min according to the revolving speed of 11000r/min, removes supernatant.Finally, with the phosphate buffer solution of 0.1M
(pH=7.4) solid sediment is redissolved to 1mL, the Proclin300 for adding 1 μ L is saved for use at 4 DEG C.
3) the fluorescent latex microballoon of rabbit igg polyclonal antibody label surface active
Take the fluorescent latex microballoon of 0.5mL surface active that 1mg carbodiimides (EDC) is added, 1mg N- hydroxysuccinimidyl acyl
Imines (NHS) stirs 4h in the revolving speed of room temperature, 120r/min, 500 μ L mg sheep rabbit igg polyclonal antibodies is then added, in room
Temperature stirs 4h under the revolving speed of 120r/min, adds 50mg BSA confining liquid, continues to stir 4h.At 2~8 DEG C, according to
The revolving speed of 11000r/min is centrifuged 30min, removes supernatant.Finally, with the phosphate buffer (pH=7.4) of 0.2M by solid
Sediment is redissolved to 1mL, and the Proclin300 for adding 1 μ L is saved for use at 4 DEG C.
4) preparation of coating pad
Use the phosphate-buffered of 0.2M molten respectively the anti-human PLGF monoclonal antibody of one plant of mouse and goat-anti rabbit polyclonal antibody
Liquid (pH=7.4) is diluted to 1mg/mL, carries out drawing film on nitrocellulose filter (NC film) with film gold spraying instrument is drawn.Contain one plant of mouse
The conduct detection line (T line) 5 of anti-human PLGF monoclonal antibody is used as nature controlling line (C line) 6 containing goat-anti rabbit polyclonal antibody,
Then at 37 DEG C, coating pad is made in dry 4h in the environment of humidity < 30%.
, 5) and the preparation of labeling pad
1. glass fibre element film is impregnated 4h in the phosphate buffer (pH=7.4) of 0.2M, then done at 35 DEG C
Dry 6h, it is spare.
2. by the fluorescent latex of the surface active for the anti-human PLGF labeling of monoclonal antibody of another plant of mouse that molar ratio is 1:0.5
Microballoon and rabbit igg label surface active fluorescent latex microballoon after mixing, according to the velocity spray of 20 μ L/cm in glass
On cellulose membrane, it is then placed at 37 DEG C dry 1h and labeling pad is made.The anti-human PLGF Dan Ke of mouse is coated in labeling pad
The place of the fluorescent latex microballoon of the surface active of the fluorescent latex microballoon and rabbit igg label of the surface active of grand antibody label
Make marks object junction 7.
6) assembling of immunochromatographydetection detection card
Then the bonding coating pad 2 first in PVC board 1 overlaps water suction in one end of the nature controlling line 6 on coating pad 2
Pad is cut into 4mm with cutting machine in one end overlap joint labeling pad 3 of the detection line 5 close to coating pad 2 and its sample pad 9 of connection
Test strips are put into getting stuck and are prepared into the detection of pregnant women placental growth factor immunochromatography by the test strips (see Fig. 1) of ± 0.1mm
Card.The structure to get stuck is the same as embodiment 1.
2. detection
The sample to be tested of 60 μ L is taken to be added drop-wise to well (the corresponding test paper of the PLGF immunochromatographydetection detection card of step 1 preparation
The sample pad 9 of item) in sample to be tested, be stored at room temperature 15min, PLGF immunochromatographydetection detection card be then inserted into fluorescence immunoassay point
Analyzer is detected, and can get testing result immediately.
3. the evaluation of test strips
1) the PLGF calibration object (0,10,40,200,1000,5000pg/mL) for configuring various concentration, with 2 step of the present embodiment
The PLGF immunochromatographydetection detection card of rapid 1 preparation is successively measured according to the detection process of step 2, and blank detection line is not higher than
10pg/mL, detection range are 10~5000pg/mL.Testing result is as shown in table 4, using concentration of specimens as abscissa, detection line with
The ratio (T/C) of nature controlling line is ordinate, it is carried out curve fitting with logistic (four parameters), degree of fitting R2Respectively
0.9984, n=6, the PLGF calibration curve of preparation as shown in figure 4, T/C value and calibration object in the model that concentration is 10-5000pg/mL
It is had good correlation in enclosing.
4 detection data of table
| Normal concentration pg/mL | 0 | 10 | 40 | 200 | 1000 | 5000 |
| T/C value | 0.1676 | 0.6355 | 0.9356 | 1.5566 | 1.9453 | 2.2654 |
2) quantitative limit
Quantitative limit (limit of quantitation, LoQ), referring to the EP17-A2 file of CLSI publication.
According to the allowable error of clinical requirement and External quality evaluation, set the overall error target of this reagent PLGF as
30%.In the case where not considering analysis deviation, allow overall error (TEa)=2CV, i.e. CV=1/2TEa.PLGF concentration exists
The CV detected when 10.00pg/mL is 12.80%, less than 15%, meets quality objective requirement, therefore LoQ=10.00pg/mL,
Show the sensitivity with higher of PLGF fluorescent microsphere immunochromatographydetection detection card.
3) precision
Extract the PLGF test strip of three batches, the sample that detectable concentration is 40,200,1000 pg/mL respectively into
Row detection, each concentration point are measured in parallel 10 times, calculate the variation within batch coefficient and batch variation of three lot number test strips
Coefficient, between criticizing as can be seen from Table 5 and variation within batch coefficient is respectively less than 14.89%, illustrates that PLGF fluorescence immune chromatography detects
The precision of card is higher.
5 detection data of table
4) rate of recovery
Taking three batch concentration is the PLGF calibration object of 5000pg/mL, is added separately to concentration close to 0 negative sample
In, wherein the volume ratio of the PLGF calibration object being added and negative sample is 1:9, replication 3 times, calculate the rate of recovery.Three batches
The result of the sample calibration product rate of recovery is respectively 93.654%, 103.96%, 95.33%, the rate of recovery 90%-110% it
Between, illustrate that measurement result complies with standard.
Embodiment 3
The preparation of 1.PLGF immunochromatographydetection detection card
1) preparation of the fluorescent latex microballoon of surface active:
The bimonthly osmanthus Ethylene Diamine diacrylate sodium of N, N'-, polyethyleneglycol moon esters of silicon acis, dodecyl benzene sulfonate and
Lauroyl glutamate, four weight ratio are 3.5:7:2:10.
1. taking surfactant (the poly- second two of the bimonthly osmanthus Ethylene Diamine diacrylate sodium of the N comprising 3.5mg, N'-, 7mg
Single month esters of silicon acis of alcohol, the dodecyl benzene sulfonate of 2mg and the lauroyl glutamate of 10mg) 0.2mol/L, pH=8.4 is added
Boric acid-borax buffer solution (PEG2000 comprising 0.5wt%~3wt%) in, add 1mL dimethylformamide, 1mg
N, N '-dicyclohexylcarbodiimide and 0.5mgN- HOSu NHS carry out anti-under the mixing speed of 120r/min
It answers；
2. taking 1mL, fluorescent latex microballoon dispersion liquid of the surface containing 1wt% with carboxyl (is purchased from Shanghai Zhen Zhun biotechnology
Co., Ltd), (include 0.5wt%~3wt%'s with boric acid-borax buffer solution of 10mL, 0.2mol/L, pH=8.4
PEG2000 after) dilution is uniformly dispersed, step is added to 1. in resulting product, it is anti-under 25 DEG C, the mixing speed of 120r/min
5h is answered, after completion of the reaction, supernatant is removed according to the revolving speed centrifugation 30min of 12000r/min, obtains the fluorescence cream of surface active
Glue microballoon, with 0.2mol/L, boric acid-borax buffer solution of pH=8.4 is redissolved spare to 1mL.
2) the fluorescent latex microballoon of the anti-human sFlt-1 labeling of monoclonal antibody surface active of another plant of mouse
1mg carbodiimides (EDC) is added in the fluorescent latex microballoon for the surface active for taking 0.5mL step 1) to prepare, 1mg
N-hydroxysuccinimide (NHS), stirs 2h under room temperature, the revolving speed of 120r/min, and it is anti-human that another plant of mouse of 300 μ L is then added
PLGF monoclonal antibody stirs 2h under room temperature, the revolving speed of 120r/min, adds 30mg BSA confining liquid, continues to stir 2h.
At 2~8 DEG C, it is centrifuged 30min according to the revolving speed of 11000r/min, removes supernatant.Finally, with the phosphate buffer solution of 0.2M
(pH=7.4) solid sediment is redissolved to 1mL, the Proclin300 for adding 1 μ L is saved for use at 4 DEG C.
3) the fluorescent latex microballoon of rabbit igg polyclonal antibody label surface active
Take the fluorescent latex microballoon of 0.5mL surface active that 1mg carbodiimides (EDC) is added, 1mg N- hydroxysuccinimidyl acyl
Imines (NHS), stirs 2h under room temperature, the revolving speed of 120r/min, and 300 μ L mg sheep rabbit igg polyclonal antibodies are then added, in
Room temperature stirs 2h under the revolving speed of 120r/min, adds 30mg BSA confining liquid, continues to stir 3h.At 2~8 DEG C, according to
The revolving speed of 11000r/min is centrifuged 30min, removes supernatant.Finally, with the phosphate buffer solution (pH=7.4) of 0.2M by solid
Sediment is redissolved to 1mL, and the Proclin300 for adding 1 μ L is saved for use at 4 DEG C.
4) preparation of coating pad
Use the phosphate-buffered of 0.01mol/L molten respectively the anti-human PLGF monoclonal antibody of mouse and goat-anti rabbit polyclonal antibody
Liquid (pH=7.4) is diluted to 1mg/mL, carries out drawing film on nitrocellulose filter (NC film) with film gold spraying instrument is drawn.It is anti-human containing mouse
The conduct detection line (T line) 5 of PLGF monoclonal antibody is used as nature controlling line (C line) 6 containing goat-anti rabbit polyclonal antibody, then
At 37 DEG C, coating pad is made in dry 4h in the environment of humidity < 30%.
5) preparation of labeling pad
1. glass fibre element film is impregnated 6h in the phosphate buffer (pH=7.4) of 0.2M, then done at 35 DEG C
Dry 8h, it is spare.
2. the fluorescent latex of the surface active for the anti-human PLGF labeling of monoclonal antibody of another plant of mouse that molar ratio is 1:2 is micro-
Ball and rabbit igg label surface active fluorescent latex microballoon after mixing, according to the velocity spray of 15 μ L/cm in glass fibers
It ties up on plain film, is then placed at 37 DEG C dry 4h and labeling pad is made.The anti-human PLGF monoclonal of mouse is coated in labeling pad
The place of the fluorescent latex microballoon of the surface active of the fluorescent latex microballoon and rabbit igg label of the surface active of antibody label is called
Marker junction 7.The purpose of setting flag object junction 7 is the same as embodiment 1.
6) assembling of immunochromatographydetection detection card
Then the bonding coating pad 2 first in PVC board 1 overlaps water suction in one end of the nature controlling line 6 on coating pad 2
Pad is cut into 4mm with cutting machine in one end overlap joint labeling pad 3 of the detection line 5 close to coating pad 2 and its sample pad 9 of connection
Test strips are put into getting stuck and are prepared into the detection of pregnant women placental growth factor immunochromatography by the test strips (see Fig. 1) of ± 0.1mm
Card.The structure to get stuck is the same as embodiment 1.
2. detection
The sample to be tested of 60 μ L is taken to be added drop-wise to well (the corresponding test paper of the PLGF immunochromatographydetection detection card of step 1 preparation
The sample pad 9 of item) in sample to be tested, be stored at room temperature 15min, PLGF immunochromatographydetection detection card be then inserted into fluorescence immunoassay point
Analyzer is detected, and can get testing result immediately.
3. the evaluation of test strips
1) the PLGF calibration object (0,10,40,200,1000,5000pg/mL) for configuring various concentration, with 3 step of the present embodiment
The PLGF immunochromatographydetection detection card of rapid 1 preparation is successively measured according to the detection process of step 2, and blank detection line is not higher than
10pg/mL, detection range are 10~5000pg/mL.Testing result is as shown in table 6, using concentration of specimens as abscissa, detection line with
The ratio (T/C) of nature controlling line is ordinate, is carried out curve fitting with logistic (four parameters), degree of fitting R2Respectively
0.9979, n=6, the PLGF calibration curve of preparation as shown in figure 5, T/C value and calibration object in the model that concentration is 10-5000pg/mL
It is had good correlation in enclosing.
6 detection data of table
| Normal concentration pg/mL | 0 | 10 | 40 | 200 | 1000 | 5000 |
| T/C value | 0.1568 | 0.6553 | 0.9535 | 1.6536 | 2.1054 | 2.3547 |
2) quantitative limit
Quantitative limit (limit of quantitation, LoQ), referring to the EP17-A2 file of CLSI publication.
According to the allowable error of clinical requirement and External quality evaluation, set the overall error target of this reagent PLGF as
30%.In the case where not considering analysis deviation, allow overall error (TEa)=2CV, i.e. CV=1/2TEa.PLGF concentration exists
The CV detected when 10.00pg/mL is 13.87%, less than 15%, meets quality objective requirement, therefore LoQ=10.00pg/mL,
Show the sensitivity with higher of PLGF fluorescent microsphere immunochromatographydetection detection card.
3) precision
The PLGF test strip of three batches is extracted, detectable concentration is the sample progress of 40,200,1000pg/mL respectively
Detection, each concentration point are measured in parallel 10 times, calculate the variation within batch coefficient and batch variation system of three lot number test strips
Number, between criticizing as can be seen from Table 7 and variation within batch coefficient is respectively less than 14.84%, illustrates PLGF fluorescence immune chromatography detection card
Precision it is higher.
7 detection data of table
4) rate of recovery
Taking three batch concentration is the PLGF calibration object of 5000pg/mL, is added separately to concentration close to 0 negative sample
In, wherein the volume ratio of the PLGF calibration object being added and negative sample is 1:9, replication 3 times, calculate the rate of recovery.Three batches
The result of the sample calibration product rate of recovery is respectively 95.86%, 103.22%, 95.77%, the rate of recovery 90%-110% it
Between, illustrate that measurement result complies with standard.
Embodiment 4
The preparation of 1.PLGF immunochromatographydetection detection card
1) preparation of the fluorescent latex microballoon of surface active:
The bimonthly osmanthus Ethylene Diamine diacrylate sodium of N, N'-, polyethyleneglycol moon esters of silicon acis, dodecyl benzene sulfonate and
Lauroyl glutamate, four weight ratio are 6:9:3:14.
1. taking the surfactant (polyethylene glycol of the bimonthly osmanthus Ethylene Diamine diacrylate sodium of the N comprising 6mg, N'-, 9mg
Single month esters of silicon acis, the dodecyl benzene sulfonate of 3mg and the lauroyl glutamate of 14mg) 0.2mol/L is added, pH=8.4's
In boric acid-borax buffer solution (PEG2000 comprising 0.5wt%~3wt%), 1mL dimethylformamide, 1mg are added
N, N '-dicyclohexylcarbodiimide and 0.5mgN- HOSu NHS, are reacted under the mixing speed of 120r/min；
2. taking 1mL, fluorescent latex microballoon dispersion liquid of the surface containing 1wt% with carboxyl (is purchased from Shanghai Zhen Zhun biotechnology
Co., Ltd), (include 0.5wt%~3wt%'s with boric acid-borax buffer solution of 10mL, 0.2mol/L, pH=8.4
PEG2000 after) dilution is uniformly dispersed, step is added to 1. in resulting product, 3h, end of reaction are stirred to react at 25 DEG C
Afterwards, according to the revolving speed centrifugation removal supernatant of 12000r/min, the fluorescent latex microballoon of surface active is obtained, with 0.2mol/L,
Boric acid-borax buffer solution of pH=8.4 is redissolved spare to 1mL.
2) the fluorescent latex microballoon of the anti-human PLGF labeling of monoclonal antibody surface active of another plant of mouse
Take the fluorescent latex microballoon of the surface active prepared in 0.5mL step 1) that 1mg carbodiimides (EDC) is added, 1mg
N-hydroxysuccinimide (NHS), stirs 3h under room temperature, the revolving speed of 120r/min, and it is anti-human that another plant of mouse of 100 μ L is then added
PLGF monoclonal antibody stirs 1h under room temperature, the revolving speed of 120r/min, adds 10mg BSA confining liquid, continues to stir 1h.
At 2~8 DEG C, it is centrifuged 30min according to the revolving speed of 11000r/min, removes supernatant.Finally, the phosphoric acid buffer with 0.01M is molten
Liquid (pH=7.4) redissolves solid sediment to 1mL, and the Proclin300 for adding 1 μ L is saved for use at 4 DEG C.
3) the fluorescent latex microballoon of rabbit igg polyclonal antibody label surface active
Take the fluorescent latex microballoon of 0.5mL surface active that 1mg carbodiimides (EDC) is added, 1mg N- hydroxysuccinimidyl acyl
Imines (NHS), stirs 3h under room temperature, the revolving speed of 120r/min, and 100 μ L mg sheep rabbit igg polyclonal antibodies are then added, in
Room temperature stirs 1h under the revolving speed of 120r/min, adds 10mg BSA confining liquid, continues to stir 1h.At 2~8 DEG C, according to
The revolving speed of 11000r/min is centrifuged 30min, removes supernatant.Finally, with the phosphate buffer (pH=7.4) of 0.2M by solid
Sediment is redissolved to 1mL, and the Proclin300 for adding 1 μ L is saved for use at 4 DEG C.
4) preparation of coating pad
Use the phosphate-buffered of 0.01mol/L molten respectively the anti-human PLGF monoclonal antibody of mouse and goat-anti rabbit polyclonal antibody
Liquid (pH=7.4) is diluted to 1mg/mL, carries out drawing film on nitrocellulose filter (NC film) with film gold spraying instrument is drawn.Contain one plant of mouse
The conduct detection line (T line) 5 of anti-human PLGF monoclonal antibody is used as nature controlling line (C line) 6 containing goat-anti rabbit polyclonal antibody,
Then at 37 DEG C, coating pad is made in dry 4h in the environment of humidity < 30%.
5) preparation of labeling pad
1. glass fibre element film is impregnated 6h in the phosphate buffer (pH=7.4) of 0.2M, then done at 35 DEG C
Dry 8h, it is spare.
2. the fluorescent latex of the surface active for the anti-human PLGF labeling of monoclonal antibody of another plant of mouse that molar ratio is 1:1 is micro-
Ball and rabbit igg label surface active fluorescent latex microballoon after mixing, according to the velocity spray of 10 μ L/cm in glass fibers
It ties up on plain film, is then placed at 45 DEG C dry 2h and labeling pad is made.Another plant of anti-human PLGF of mouse is coated in labeling pad
The ground of the fluorescent latex microballoon of the surface active of fluorescent latex microballoon and the rabbit igg label of the surface active of labeling of monoclonal antibody
Side is called marker junction 7.The purpose of setting flag object junction 7 is the same as embodiment 1.
6) assembling of immunochromatographydetection detection card
Then the first bonding coating pad 2 in PVC board 1 overlaps water absorption pad in one end of the nature controlling line 6 on coating pad 2,
Close to coating pad 2 detection line 5 one end overlap joint labeling pad 3 and its connection sample pad 9, with cutting machine be cut into 4mm ±
The test strips (see Fig. 1) of 0.1mm, test strips are put into getting stuck and are prepared into pregnant women placental growth factor immunochromatographydetection detection card.
The structure to get stuck is the same as embodiment 1.
2. detection
The sample to be tested of 60 μ L is taken to be added drop-wise to well (the corresponding test paper of the PLGF immunochromatographydetection detection card of step 1 preparation
The sample pad 9 of item) in sample to be tested, be stored at room temperature 15min, PLGF immunochromatographydetection detection card be then inserted into fluorescence immunoassay point
Analyzer is detected, and can get testing result immediately.
3. the evaluation of test strips
1) the PLGF calibration object (0,10,40,200,1000,5000pg/mL) for configuring various concentration, with 4 step of the present embodiment
The immunochromatographydetection detection card of rapid 1 preparation is measured according to the detection process of step 2, and blank detection line is not higher than 10pg/mL, inspection
Survey range is 10~5000pg/mL.Testing result is as shown in table 8, using concentration of specimens as abscissa, the ratio of detection line and nature controlling line
Being worth (T/C) is ordinate, is carried out curve fitting with logistic (four parameters), and degree of fitting R2 is respectively 0.9986, n=6, preparation
PLGF calibration curve as shown in fig. 6, T/C value and calibration object have good phase in the range of concentration is 10-5000pg/mL
Guan Xing.
8 detection data of table
| Normal concentration pg/mL | 0 | 10 | 40 | 200 | 1000 | 5000 |
| T/C value | 0.1645 | 0.5789 | 0.8956 | 1.5745 | 2.0764 | 2.2654 |
2) quantitative limit
Quantitative limit (limit of quantitation, LoQ), referring to the EP17-A2 file of CLSI publication.
According to the allowable error of clinical requirement and External quality evaluation, set the overall error target of this reagent PLGF as
30%.In the case where not considering analysis deviation, allow overall error (TEa)=2CV, i.e. CV=1/2TEa.PLGF concentration exists
The CV detected when 10.00pg/mL is 14.86%, less than 15%, meets quality objective requirement, therefore LoQ=10.00pg/mL,
Show the sensitivity with higher of PLGF fluorescent microsphere immunochromatographydetection detection card.
3) precision
The PLGF test strip of three batches is extracted, detectable concentration is the sample progress of 40,200,1000pg/mL respectively
Detection, each concentration point are measured in parallel 10 times, calculate the variation within batch coefficient and batch variation system of three lot number test strips
Number, between criticizing as can be seen from Table 9 and variation within batch coefficient is respectively less than 14.67%, illustrates PLGF fluorescence immune chromatography detection card
Precision it is higher.
9 detection data of table
4) rate of recovery
Taking three batch concentration is the PLGF calibration object of 5000pg/mL, is added separately to concentration close to 0 negative sample
In, wherein the volume ratio of the PLGF calibration object being added and negative sample is 1:9, replication 3 times, calculate the rate of recovery.Three batches
The result of the sample calibration product rate of recovery is respectively 97.97%, 105.98%, 98.92%, the rate of recovery 90%-110% it
Between, illustrate that measurement result complies with standard.
It is attached: required solution allocation
(1) 0.2M phosphate buffer solution
NaH2PO4.H2O 5.24g；
Na2HPO4.2H2O 28.83g；
Purified water is settled to 1000mL；
(2) boric acid-borax buffer solution
Borax 19.07g；
Boric acid 12.37g；
5~30g of PEG2000；
Purified water is settled to 1000mL.
Embodiment described above be only for absolutely prove the present invention and for embodiment, protection scope of the present invention is unlimited
In this.Those skilled in the art's made equivalent substitute or transformation on the basis of the present invention, in protection of the invention
Within the scope of.Protection scope of the present invention is subject to claims.
Claims (10)
1. one kind quickly detects the immunochromatographydetection detection card of pregnant women placental growth factor, including test strips, the test strips include
Detection line and nature controlling line, which is characterized in that the anti-human PLGF monoclonal antibody of one plant of mouse, the Quality Control are coated in the detection line
Goat-anti rabbit polyclonal antibody is coated on line.
2. the immunochromatographydetection detection card of quickly detection pregnant women placental growth factor as described in claim 1, which is characterized in that institute
Stating test strips further includes PVC board, and sequentially connected sample pad, labeling pad, coating pad and water suction are fixed in the PVC board
Pad, the packet, which is covered with, is successively arranged detection line and nature controlling line, and the sample pad and labeling pad are connected as one.
3. the immunochromatographydetection detection card of quickly detection pregnant women placental growth factor as claimed in claim 2, which is characterized in that institute
It states coating pad and is connected with labeling pad close to one end of detection line, one end close to nature controlling line is connected with water absorption pad.
4. the immunochromatographydetection detection card of quickly detection pregnant women placental growth factor as claimed in claim 3, which is characterized in that institute
State the fluorescent latex microballoon that the surface active of the anti-human PLGF labeling of monoclonal antibody of another plant of mouse is coated in labeling pad and rabbit
The fluorescent latex microballoon of the surface active of IgG label, the surface active of the anti-human PLGF labeling of monoclonal antibody of another plant of mouse
Fluorescent latex microballoon and rabbit igg label surface active fluorescent latex microballoon molar ratio be 1:0.2~4.
5. the immunochromatographydetection detection card of quickly detection pregnant women placental growth factor as claimed in claim 4, which is characterized in that institute
The immunochromatographydetection detection card for stating quickly detection pregnant women placental growth factor further includes that getting stuck for test strips is set for card.
6. the immunochromatographydetection detection card of quickly detection pregnant women placental growth factor as claimed in claim 5, which is characterized in that institute
It states to get stuck and includes:
Kerve is connected to the PVC board；
Upper cover is connected to the kerve, the well for being loaded in the sample pad is provided on the upper lid；
Observation window is set on lid and for detection line and the acquisition of the data of nature controlling line.
7. a kind of system of the immunochromatographydetection detection card of quick detection pregnant women placental growth factor described in any one of claims 1-6
Preparation Method, which comprises the following steps:
1) preparation of coating pad: the anti-human PLGF monoclonal antibody of one plant of mouse and goat-anti rabbit polyclonal antibody are coated with respectively to nitric acid
On cellulose membrane, drying for standby；
2) preparation of labeling pad: by the fluorescent latex microballoon of the surface active of the anti-human PLGF labeling of monoclonal antibody of another plant of mouse
After the fluorescent latex microballoon mixing of the surface active of rabbit igg label, it is sprayed on glass fibre element film, drying for standby；
3) test strips are assembled: the bonding coating pad in PVC board, and in the overlap joint water suction of the one end for the nature controlling line being covered with close to the packet
Pad, overlap joint labeling pad and its sample pad of connection in the one end for the detection line being covered with close to the packet；Then it is cut into institute
The test strips of width are needed, the reagent strip is put into gets stuck later.
8. preparation method as claimed in claim 7, which is characterized in that the fluorescent latex microballoon of the surface active passes through following
Step is made:
1) take surfactant be added 0.1~0.5mol/L, pH value be 8~10 boric acid-borax buffer solution in, add two
Methylformamide, N, N '-dicyclohexylcarbodiimide and n-hydroxysuccinimide, are stirred to react；The boric acid-borax buffering
Solution includes the PEG2000 of 0.5wt%~3wt%；
2) take surface with the dispersion liquid of the fluorescent latex microballoon of carboxyl, after boric acid-borax buffer solution tune pH to 8~10,
It is added in the resulting product of step 1), 1~5h is stirred to react at 25 DEG C, after completion of the reaction, centrifugation removal supernatant obtains
The fluorescent latex microballoon of surface active is redissolved spare with boric acid-borax buffer solution；Boric acid-the borax buffer solution includes
The PEG2000 of 0.5wt%~3wt%.
9. preparation method as claimed in claim 8, which is characterized in that the surfactant includes N, the bis- lauroyl of N'-
Base ethylenediamine diacrylate sodium, polyethyleneglycol moon esters of silicon acis, dodecyl benzene sulfonate and lauroyl glutamate, four
Weight ratio is (0.5~6): (4~9): (0.8~3): (5~14)；The fluorescence of the surfactant and amino surface cream
The weight ratio of glue microballoon is (0.5~120): 1.
10. preparation method as claimed in claim 8 or 9, which is characterized in that the anti-human PLGF monoclonal antibody of another plant of mouse
The fluorescent latex microballoon of the surface active of label is made by following steps:
It takes the fluorescent latex microballoon of surface active to be added in carbodiimides and n-hydroxysuccinimide and 2~6h is stirred at room temperature,
Then the anti-human PLGF monoclonal antibody of another plant of mouse is added, stirs 1~4h at room temperature, adds 10~50mg BSA confining liquid,
Continue 1~4h of stirring；At 2~8 DEG C, it is centrifuged 30min according to the revolving speed of 11000r/min, removes supernatant；Finally, with
The phosphate buffer solution of 0.01M~0.5M, pH=7.4 redissolve solid sediment, add Proclin300 and save at 4 DEG C
For use；
Wherein, the fluorescent latex microballoon of the surface active and the mass ratio of the anti-human PLGF monoclonal antibody of another plant of mouse are 1:
(0.01~4).
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| CN112557669A (en) * | 2020-09-30 | 2021-03-26 | 杭州华视诺维医疗科技有限公司 | Kit and application thereof in detection of vascular endothelial growth factor in anterior chamber fluid |
| CN112698042A (en) * | 2020-12-17 | 2021-04-23 | 北京赛诺浦生物技术有限公司 | Fluorescent immunochromatography test strip for detecting human growth differentiation factor-15 and preparation method and application thereof |
| CN113804899A (en) * | 2021-08-26 | 2021-12-17 | 宁波奥丞生物科技有限公司 | Immunochromatography reagent strip for detecting urine placenta growth factor of pregnant woman and preparation method thereof |
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Application publication date: 20191129 |