阅读说明：本技术 一种快速检测孕妇胎盘生长因子的免疫层析检测卡及其制备方法 (A kind of immunochromatographydetection detection card and preparation method thereof of quick detection pregnant women placental growth factor ) 是由 林斯 张衡 肖燚 于 2019-09-26 设计创作，主要内容包括：本发明涉及一种快速检测孕妇胎盘生长因子的免疫层析检测卡,包括试纸条,包括试纸条,所述试纸条包括检测线及质控线,所述检测线上包被有一株鼠抗人PLGF单克隆抗体,所述质控线上包被有羊抗兔多克隆抗体。本发明所提供的快速检测孕妇胎盘生长因子的免疫层析检测卡,其可用于血清、血浆和全血的检测,以用于预测子痫前期相关疾病的诊断。(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.)

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).

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.