CN206725584U - The tumor marker rapid detection card of double antibody nanogold particle mark - Google Patents

Abstract

The utility model provides a kind of tumor marker rapid detection card, the detection is stuck in the nano antibody that antitumor label CEA and p53 mutant are coated with immune response area nitrocellulose filter, and the anti-CEA and p53 mutant secondary antibody of marking nano gold grain is coated with upstream, using double-antibody method immunochromatography Cleaning Principle, the sample from human body fluid can be carried out to detect whether containing tumor marker CEA and p53 mutant.Methods described is easy to operate, visual detection can be directly carried out, high sensitivity, high specificity, CEA and P53 mutant can be detected simultaneously, the association of two kinds of antigen can play cooperative compensating effect together, and auxiliary judgment is provided for clinical tumor early diagnosis.

Description

The tumor marker rapid detection card of double antibody nanogold particle mark

Technical field

The utility model discloses a kind of tumor marker rapid detection card, belongs to the outer field of fast detection of aleuroplast.

Background technology

Carcinomebryonic antigen (CEA, also known as CEACAM-5 or CD66e) is a kind of glycoprotein with about 180kDa molecular weight. CEA is a member of immunoglobulin superfamily, and contains and be connected via glycosyl-phosphatidyl inositol (GPI) anchor with cell membrane 7 domains.7 domains include single N-terminal Ig variable domains and 6 domains (A1-B1-A2-B2-A3-B3) homologous with Ig constant domains. CEA primary classifications are the protein only expressed in fetal tissue, are identified now in several normal adult tissues. CEA overexpression is observed in the cancer of many types, including colorectal cancer, cancer of pancreas, lung cancer, stomach cancer, liver cell Knurl, breast cancer and thyroid cancer.Therefore, CEA has been identified as tumor associated antigen.CEA is easily cut from cell surface, And directly or via lymphatic system from tumour comes off blood flow.Due to this characteristic, the horizontal as facing of change of serum C EA is used Bed mark is to diagnose cancer and screen cancer.Moreover, CEA has also been used as tumor marker, measures and risen in cancer patient's blood High CEA Radioimmunoassay of vascular endothelial growth has been used clinically for the prognosis and control of cancer.

P53 genes are a kind of tumor suppressor genes, are positioned at human chromosomal 17p13.1, encode what 393 amino acid formed Phosphorylated protein in 53kD core, it is referred to as p53 albumen.P53 genes are the negative growth factors in cell growth cycle, with cell The important biological function such as the regulation and control in cycle, DNA reparations, cell differentiation, Apoptosis is relevant.P53 genes are divided into wild type With two kinds of saltant type, its product also has wild type and saltant type.Wild-type p 53 protein is extremely unstable, only several minutes of half-life period, and Tumor inhibition effect with trans-activation function and wide spectrum.The increase of mutant p53 protein stability, Increased Plasma Half-life can quilt ImmunohistochemistryMethods Methods detect.The mutation (missing) of p53 genes is the frequent event of human tumor, generation, hair with tumour Open up relevant.It is generally acknowledged that p53 overexpressions are related to the transfer, recurrence and poor prognosis of tumour.

The carly fruit drop of the tumour of wide spectrum in the market only relies solely on the detection of single tumor markers, often Round of visits length single Chang Yinwei, sensitivity is low, so as to can not ensure the diagnosis of tumour, or even after influenceing tumor patient Continuous treatment and life and health.

Nanogold refers to the molecule of gold, and its diameter has high electron density, dielectric property and catalysis in 1~100nm Effect, can be combined, and do not influence its bioactivity with a variety of large biological molecules.Nano gold mark technology (nanogold Labelling techique), substantially the macromolecule such as protein is adsorbed to the coating process on nanogold particle surface.Inhale Random reason is nanogold particle surface negative charge, and strong bonded is formed because of Electrostatic Absorption with the positive charge group of protein, and And will not be denatured biomolecule after adsorbing, and because gold grain has the characteristic of high electron density, at gold mark protein binding, Visible dark brown coloured particles under microscope, when these labels are largely assembled at corresponding part, naked eyes red color visible or powder Punctation, thus in qualitative or sxemiquantitative tachysynthesis detection method.Because spherical nano Au particle is to albumen Matter has very strong adsorption function, can with staphylococcal protein A, immunoglobulin, toxin, glycoprotein, enzyme, antibiotic, hormone, The Non-covalent bindings such as bovine serum albumin(BSA), thus turn into highly useful instrument in basic research and experiment.Immunochromatographic method (gold immunochromatography assay, GICA) is that various reaction reagents are fixed on into same test paper with ribbon On bar, sample to be checked is added in one end of test strips, after a kind of reagent is dissolved, is percolated, moved on chromatography strip by capillarity Row simultaneously contacted with another reagent on film, the determinand in sample with chromatographic material be directed to determinand acceptor (such as antigen or Antibody) specific immune response occurs.Immune complex is trapped, is gathered in the certain area of chromatographic material in chromatography process (detection band), intuitively developed the color result by the nano gold mark thing that can be estimated.And free label then crosses detection band, Reach the purpose being automatically separated with binding label.GICA features are single agents, and single stepping, whole reagents can be in room temperature It is long-term to preserve.Nanogold immune detection assay is advanced to a brand-new stage by this new method.

Therefore, the purpose of this utility model is to provide a kind of easy to operate, high sensitivity based on biomarker, special Property it is strong, the tumour early detection card of CEA and p53 mutant can be detected simultaneously.

Utility model content

Based on above-mentioned purpose, the utility model provides a kind of tumor marker rapid detection card, the detection card by The base material 1 of list structure provides support, and coated film 2 is set on the base material 1, on the coated film 2 by from left to right according to It is secondary to be provided with sample application zone 3, detection zone 4 and suction zones 5, nanogold coupling pad 31 is overlapped on the sample application zone 3, in nanogold coupling Close and sample pad 32 is overlapped on pad 31, blotting paper 51 is overlapped on the suction zones 5, series connection row is provided with the detection zone 4 Detection line 1, detection line 2 42 and the nature controlling line 43 of row.

In a preferred embodiment, the coated film 2 is nitrocellulose filter, and pad 31 is coupled in the nanogold It is upper to be coated with anti-CEA secondary antibodies, anti-p53 mutant secondary antibody and the rabbit igg antibody coupled with nanogold, in the detection line Anti- CEA first antibodies are coated with one 41, anti-p53 mutant first antibody are coated with the detection line 2 42, in the Quality Control Goat anti-rabbit igg antibody is coated with line 43.

Preferably, the CEA antibodie, anti-p53 mutant antibodies are monoclonal antibody.

Preferably, the CEA antibodie, anti-p53 mutant antibodies are nano antibody.

In a preferred embodiment, the detection line 1, detection line 2 42 and nature controlling line 43 at intervals of 2mm-5mm。

In a preferred embodiment, the base material 1 is made up of PVC material.

Preferably, cutting ferrule 6 is further equipped with the periphery of the base material 1, it is open in the front of cutting ferrule 6 to coat base material 1 There are the detection zone form 61 being rectangle and rounded well 62, so that detection line 1, detection line two are left in sample application zone 42 and nature controlling line 43 do not coated by cutting ferrule 6.

It is further preferable that a diameter of 2mm-10mm of the well 62.

The tumor marker rapid detection card of double antibody nanogold particle provided by the utility model mark is easy to operate, can Directly to carry out visual detection, high sensitivity, high specificity, the tumour early stage that can detect CEA and p53 mutant simultaneously examines Card to be surveyed, auxiliary judgment is provided for tumour early stage, the association of two kinds of antigen can play cooperative compensating effect together, Detection time is shortened, adds sensitivity and the specificity of detection.

Brief description of the drawings

Fig. 1 tumor marker rapid detection card three-dimensional exploded views；

Fig. 2 tumor marker rapid detection card sides position profile；

Fig. 3 tumor marker rapid detection card testing result top views

Embodiment

The utility model is further described with reference to specific embodiment, and the advantages of the utility model and feature will be with Description and it is apparent.But these embodiments are only exemplary, the scope of protection of the utility model are not formed any Limitation.

Embodiment

Fig. 1 is the three-dimensional exploded view of rapid detection card one specific embodiment of the utility model based on tumor marker； The detection card sets the coating of nitrocellulose filter by providing support in the PVC base 1 of list structure on the base material 1 Film 2.By being disposed with sample application zone 3, detection zone 4 and suction zones 5 from left to right on the nitrocellulose filter, add described Nanogold coupling pad 31 is overlapped in sample area 3, sample pad 32, nanogold coupling pad 31 and sample are overlapped on nanogold coupling pad 31 Pad 32 selects glass fibre or polyester material.Blotting paper 51 is overlapped with the suction zones 5, is set on the detection zone 4 There are the detection line 1, detection line 2 42 and nature controlling line 43 of arranged in series；The detection line 1, detection line 2 42 and Quality Control Line 43 at intervals of 2mm-5mm.It is described have be coated with the anti-CEA second that couple with nanogold on nanogold coupling pad 31 and resist Body, anti-p53 mutant secondary antibody and rabbit igg antibody, are coated with anti-CEA first antibodies, in institute in the detection line 1 State and anti-p53 mutant first antibody is coated with detection line 2 42, goat anti-rabbit igg antibody is coated with the nature controlling line 43； The CEA antibodie, anti-p53 mutant antibodies are nano antibody.

Fig. 2 is the side position profile of the utility model one specific embodiment of the rapid detection card based on tumor marker. PVC base 1, coated film 2 (nitrocellulose filter), nanogold coupling pad 31, sample pad 32, blotting paper are followed successively by from lower to upper 51。

Fig. 3 is that the testing result of the utility model one specific embodiment of the rapid detection card based on tumor marker is overlooked Figure.In the periphery of the base material 1 equipped with cutting ferrule 6, to coat base material 1, being opened in the front of cutting ferrule 6 has the inspection being rectangle Area's form 61 and rounded well 62 are surveyed, so that detection line 1, detection line 2 42 and nature controlling line 43 are left not in sample application zone Coated by cutting ferrule 6.

The preparation method of the utility model tumor marker rapid detection card

1) preparation of sample pad：Sample pad selects glass fibre, is dissolved with buffer salt (PBS, Tris or glycine etc.) etc. 5mg/ml albumen (BSA, casein etc.), a small amount of surfactant (Tween20 etc.) is then added, adjusts pH to 6~8, Glass fiber or polyester material are placed in immersion 2~4 hours, after taking-up 25 DEG C of dryings can obtain within 8 hours.

2) preparation of nanogold coupling pad：The anti-CEA of nano gold mark will be equably sprayed in the sample pad handled well Two antibody, anti-p53 mutant secondary antibody and rabbit igg antibody, being placed in 25 DEG C of dryings can obtain for 4 hours.

3) processing of nitrocellulose filter：

A. the preparation of detection line 41：Anti- CEA nano antibodies are diluted to 2.0mg/ml with 20Mm pH7.2 PB buffer solutions Concentration, 0.8ul/cm rule on nitrocellulose filter and obtains detection line 41, and nitrocellulose filter 5 then is placed in into drying box Interior 20 DEG C of forced air drying 12h are produced；

B. the preparation of detection line 42：Anti- p53 mutant nano antibody is diluted to 20Mm pH7.2 PB buffer solutions 2.0mg/ml concentration, 0.8ul/cm rule on nitrocellulose filter and obtain detection line 52, then put nitrocellulose filter 5 Produced in 20 DEG C of forced air drying 12h in drying box；

C. the preparation of nature controlling line 43：Goat anti-rabbit igg antibody is pressed to 4mg/ml concentration, 0.8ul/cm is in nitrocellulose Rule on film to obtain nature controlling line 43, detection line 41,42 is parallel with nature controlling line 43, is spaced 5mm, drying condition is the same as detection line 41,42；

D. nitrocellulose filter is closed with bovine serum albumin(BSA).

4) preparation of adsorptive pads：It is suitable for from conventional be cut into blotting paper in this area on base material and fine with nitric acid Tie up the size of plain film overlap joint.

5) assemble：Sample pad and blotting paper are part generally in the art.By above-mentioned nitrocellulose filter, adsorptive pads, sample Product pad is pasted onto in PVC base, and puts the adaptable cutting ferrule 6 of size.

The utility model tumor marker quick detection card test method

Blotting paper 51 contains cellulose, the larger molecular organicses are crisscross in netted, therefore cellulose is staggered to form There are many spaces in netted, these spaces can contain the moisture on blotting paper 51, so as to produce tension force.And because concentration is produced Raw potential energy, sample is flowed to the low side of concentration from the high side of concentration, therefore in experimentation, blotting paper 51 is put in The offside of sample pad 32,100 μ L are added dropwise in the detection sample that in sample pad 32, can promote to be added dropwise in detected serum, whole blood Quickly spread to nanogold coupling pad and nitrocellulose filter at sample-adding.Nitrocellulose filter is the point of immune response. Padded when the sample containing CEA and p53 mutant antigens diffuses to nanogold coupling first, herein with being marked with nano Au particle Specific immune response occurs for anti-CEA secondary antibodies, anti-p53 mutant secondary antibody, produces the immune compound of nano gold mark Thing, continue under tension force effect caused by blotting paper to nitrocellulose membrane diffusion, at detection line 41, nano gold mark resists With the CEA antibodie at this immune occurs again for CEA secondary antibodies/CEA immune complexs, forms the immune of double antibodies sandwich Compound, and show red line, then detected for CEA positive；At detection line 42, the anti-p53 mutant of nano gold mark With the anti-p53 mutant antibodies at this immune response occurs again for two antibody/p53 mutant immune complex, forms dual anti-folder The immune complex of the heart, and show red line, then detected for p53 mutant positive；At nature controlling line 43, coupled through nanogold Pad the nano gold mark rabbit igg antibody diffused to and immune response occurs with the goat anti-rabbit igg antibody at this, formed immune compound Thing, and show red line, then normal for system response, credible result.

It can be 5-10min that above immune response is totally time-consuming；After reaction terminates, according to being at detection line in form 41 and 42 It is no red line judged result positive or negative occur, whether occur red line at nature controlling line and judge whether system is working properly, result is It is no credible.

Claims (8)

1. a kind of tumor marker rapid detection card, it is characterised in that the detection card is provided by the base material (1) in list structure Support, coated film (2) is set on the base material (1), by being disposed with sample application zone from left to right on the coated film (2) (3), detection zone (4) and suction zones (5), nanogold coupling pad (31) is overlapped on the sample application zone (3), couples and pad in nanogold (31) sample pad (32) is overlapped on, blotting paper (51) is overlapped on the suction zones (5), is provided with the detection zone (4) Detection line one (41), detection line two (42) and the nature controlling line (43) of arranged in series.

2. detection card according to claim 1, it is characterised in that the coated film (2) is nitrocellulose filter, described Anti- CEA secondary antibodies, anti-p53 mutant secondary antibody and the rabbit igg coupled with nanogold is coated with nanogold coupling pad (31) Antibody, anti-CEA first antibodies are coated with the detection line one (41), anti-p53 mutation are coated with the detection line two (42) Body first antibody, goat anti-rabbit igg antibody is coated with the nature controlling line (43).

3. detection card according to claim 2, it is characterised in that the CEA antibodie, anti-p53 mutant antibodies are single Clonal antibody.

4. detection card according to claim 2, it is characterised in that the CEA antibodie, anti-p53 mutant antibodies are to receive Meter Kang Ti.

5. detection card according to claim 2, it is characterised in that the detection line one (41), detection line two (42) and matter Control line (43) at intervals of 2mm-5mm.

6. detection card according to claim 1, it is characterised in that the base material (1) is made up of PVC material.

7. detection card according to claim 6, it is characterised in that be further equipped with cutting ferrule in the periphery of the base material (1) (6), to coat base material (1), there is the detection zone form (61) that is rectangle and rounded add the cutting ferrule (6) front is open Sample hole (62), so that sample application zone is left detection line one (41), detection line two (42) and nature controlling line (43) and do not wrapped by cutting ferrule (6) Cover.

8. detection card according to claim 7, it is characterised in that a diameter of 2mm-10mm of the well (62).