Saliva cortisol rapid immunochromatography strip as well as preparation method and application thereof

Abstract

The invention provides a saliva cortisol rapid immunochromatography strip as well as a preparation method and application thereof. The saliva cortisol rapid immunochromatography strip comprises a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper which are sequentially overlapped on a PVC (polyvinyl chloride) bottom plate, and a colloidal gold labeled cortisol monoclonal antibody and colloidal gold labeled IgY (Immunoglobulin Y) are sprayed on the combination pad. The rapid saliva cortisol immunochromatography strip based on the colloidal gold probe is used for in-vitro detection and has important significance on Cushing syndrome diagnosis and healing monitoring. Meanwhile, the method has certain indication significance on stress, depression and other mental health diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medical devices, and particularly relates to a saliva cortisol rapid immunochromatographic strip, a preparation method and application thereof. BACKGROUND

[0002] Cortisol, molecular formula is C 21 H 30 O5, also known as hydrocortisone, is a glucocorticoid mainly secreted by the adrenal gland. In the peripheral blood, cortisol is mainly transported in combination with corticosteroid-binding globulin and albumin, and only 3%-5% exists in free form and has biological activity. Free cortisol can freely diffuse through the cell membrane into saliva due to its liposolubility, thereby rapidly maintaining balance in blood and saliva. The cortisol secreted by the human body every day has circadian rhythm changes, and the secretion amount is usually the highest in the morning and the lowest at midnight. Midnight is the best time point for sensitive diagnosis of Cushing's syndrome. The cortisol in saliva, as liposoluble free cortisol, has biological activity and can more directly reflect the disease state. The saliva cortisol is not affected by changes in serum corticosteroid-binding globulin, saliva flow rate, age, gender, body mass index, whether smoking, and disease duration. Accurate determination of the content of cortisol has important significance for the clinical diagnosis of diseases related to cortisol. In clinical practice, the determination of cortisol level is often used as a laboratory diagnostic method for adrenal-related endocrine diseases such as Cushing's syndrome, acromegaly, short stature, abnormal sex hormone levels, etc. In addition, cortisol is sensitive to stress, and the level will increase under conditions such as emotional tension, injury, fatigue, illness, high training load, etc., and thus can also be used to evaluate the physiological and psychological stress state.

[0003] The content of cortisol in saliva is very low, and the critical value for midnight diagnosis is only 1.45 ng / mL. In addition, the composition of saliva is complex, and there are many exogenous interference factors, so the sensitivity requirement of the detection method is very high. At present, the detection methods of saliva cortisol include immunoassay (such as radioimmunoassay, chemiluminescence immunoassay, enzyme-linked immunosorbent assay), liquid chromatography tandem mass spectrometry (LC-MS / MS), gas chromatography-mass spectrometry and electrochemical method, etc. However, these methods are relatively complex to operate, and usually require large instruments to complete the detection. SUMMARY

[0004] Therefore, the purpose of the present application is to overcome the defects in the prior art, and to provide a saliva cortisol rapid immunochromatographic strip, a preparation method and application thereof. The saliva cortisol rapid immunochromatographic strip based on the colloidal gold probe of the present application is used for in vitro detection, and has important significance for Cushing's syndrome diagnosis and healing monitoring. At the same time, it has certain indicative significance for psychological health diseases such as stress and depression.

[0005] Before setting forth the invention, definitions of terms used in the specification are provided below:

[0006] The term "PVC" refers to: Polyvinyl chloride.

[0007] The term "NC membrane" refers to: Nitrocellulose membrane.

[0008] The term "IgY" refers to: A class of antibodies found universally in birds, reptiles and amphibians, consisting of two heavy chains (H) and 2 light chains (L), in which the heavy chain (υ) contains 4 constant regions, but does not contain the hinge region, and the isoelectric point is close to 5.2.

[0009] The term "gold micro-nano microspheres" refers to: A kind of nanometer microspheres material with particle size range of 150-450 nm composed of micro gold particles and nanometer materials by direct physical adsorption process, typical manufacturers include but are not limited to Zhengzhou Lingsi Biological Technology Co., Ltd.

[0010] The term "quality control C line" refers to: A line with quality control marks, used to verify the effectiveness of the reagent card and the correctness of the operation process, when the C line appears, it means that the reagent card and the operation method are correct, and the result of the T line can be determined.

[0011] The term "detection T line" refers to: Used to determine whether the target cortisol exists in the sample. If the T line appears, it means that the sample does not contain the target cortisol, and the result is negative; If the T line does not appear, it means that the sample contains the target cortisol, and the result is positive.

[0012] The term "competitive method" refers to: The competitive method is usually used to detect small molecules such as haptens, hormones, drugs, etc. The immune test paper detection platform using the competitive method, the label antibody is fixed on the conjugate pad of the reagent strip, and the T line on the NC membrane is fixed with the large molecule coupled to be detected antigen (small molecule antigen cannot be directly fixed on the NC membrane, therefore, it is necessary to couple the small molecule to the BSA etc. Large molecule substances by chemical method and then fixed on the NC membrane), and the secondary antibody is fixed on the C line. The method is just the opposite of the conventional method to judge the result.

[0013] The term "PBS" refers to: Phosphate buffer.

[0014] The term "PEG" refers to: Polyethylene glycol.

[0015] The term "BSA" refers to: Bovine serum albumin.

[0016] The term "Proclin 300" refers to: a liquid biocide, ProClin preservative for IVD production, is a mixture of two isothiazolinones, containing 5-Chloro-2-methyl-4-isothiazolin-3-one (CMIT) and 2-Methyl-4-isothiazolin-3-one (MIT) in a ratio of 3:1, the content of this mixture is 3.0-3.6%.

[0017] The term "Proclin 950" refers to: ProClin preservative for IVD production, the main active ingredient is a 9.5% solution of 2-methyl-4-isothiazolin-3-one.

[0018] The term "KroVin 300" refers to: a broad-spectrum, low-toxicity, environmentally friendly preservative, the bacteriostatic active ingredient is CMIT / MIT (chloromethyl isothiazolinone / methyl isothiazolinone), the product composition contains alkyl carboxylate and solvent (modified glycol).

[0019] The term "KroVin 950" refers to: a stable, low-toxicity, environmentally friendly preservative, the active ingredient is 2-methyl-4-isothiazolin-3-one (MIT).

[0020] The term "Rozone S16" refers to: a composite bactericidal and bacteriostatic preservative, the Chinese name is: broad-spectrum high-efficiency preservative Rozone S16, its main component is usually isothiazolinone compounds, one of the common components is 2-methyl-4-isothiazolinone (Methylisothiazolinone, MI).

[0021] The term "PEG6000" refers to: polyethylene glycol PEG6000 is a high molecular weight polymer condensed from ethylene oxide and water, it is a milky white solid, the hydroxyl value is between 17.5-20, the molecular weight is between 5500-7000, it is easily soluble in water or ethanol, and insoluble in diethyl ether. Moreover, it is a non-toxic, non-irritating, widely used in various pharmaceutical preparations.

[0022] The term "PEG20000" refers to: is a polyethylene glycol with an average molecular weight of 20,000, soluble in water, and many polar solvents such as acetone, ethanol and chlorinated solvents. PEG aqueous solution is stable at room temperature, the first use is recommended to be filtered with a 0.45 μm filter to remove impurities.

[0023] The term "PVP-3000" refers to: polyvinylpyrrolidone with a molecular weight of 3000. White or near-white or slightly yellowish protein-like solid. Slightly odorous. PVP is easily soluble in water, alcohol, amines and halogenated hydrocarbons, insoluble in acetone, diethyl ether, etc. It has excellent solubility, biocompatibility, physiological inertness, film-forming property, film body protection ability and the ability to complex with a variety of organic and inorganic compounds. It is relatively stable to acids, salts and heat, so it has a wide range of uses.

[0024] The term "DPBS" refers to: Dulbecco's phosphate buffered saline, DPBS differs from PBS in that it also includes potassium chloride.

[0025] The term "surfactant S6" refers to: the chemical name is BIO-TERGE AS-40, the CAS number is 68439-57-6, and the molecular weight is 315. It belongs to anionic surfactants, has mild surface activity, and has good compatibility with solvents. In the development process of colloidal gold / latices rapid diagnosis test strip products, S6 can be used as a mild surfactant to help solve the wetting and solubility problems of urine test paper, thereby improving accuracy, accelerating sample wetting, improving reaction speed, and enhancing enzyme stability and activity.

[0026] The term "surfactant S9" refers to: a propylene oxide-ethylene oxide-vinyl dimethyl amine copolymer, also known as Tetronic 1307, which belongs to a zwitterionic surfactant. It has very high hydrophilicity, with an HLB value greater than 24, and is a water-soluble surfactant in the field of immunochromatography. S9 has good interfacial activity, combining the advantages of anionic and cationic surfactants, and has no cloud point phenomenon and is not easily affected by inorganic electrolytes. In addition, it also has excellent emulsifying ability, dispersing ability and antistatic effect.

[0027] The term "surfactant S17" refers to: an anionic surfactant with the chemical name sodium heptadecyl sulfonate or sodium heptadecyl sulfate. It is a white or slightly yellow solid with good solubility and can form emulsions or micelles in water, exhibiting good surface activity and interfacial activity. S17 has strong stain removal, emulsification, moisturizing and defoaming properties, as well as certain antistatic properties and stability.

[0028] The term "surfactant S21" refers to a non-ionic surfactant, also known as Brij 35. Its chemical name is polyoxyethylene lauryl ether, with the following properties: CAS No. 9043-30-5, molecular weight: about 1200, HLB value: 16.9, physical form: white waxy solid at room temperature, main functions: good emulsification, solubilization, dispersion, wetting, etc. Application fields: commonly used in detergents, shampoo (including baby shampoo), facial cleanser, bath liquid, cosmetics, etc. It can improve the emulsion stability and cleaning effect of the product, and has low skin irritation.

[0029] To achieve the above-mentioned purpose, the first aspect of the present application provides a saliva cortisol rapid immunochromatographic strip, comprising: a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent paper sequentially lapped on a PVC base plate; wherein:

[0030] The conjugate pad is sprayed with nanoparticle-labeled cortisol monoclonal antibody and nanoparticle-labeled IgY, preferably selected from one or more of the following: avian IgY, reptile IgY, amphibian IgY, wherein the avian IgY is preferably selected from one or more of the following: chicken IgY, duck IgY, goose IgY, most preferably chicken IgY.

[0031] According to the saliva cortisol rapid immunochromatographic strip of the first aspect of the present application, wherein,

[0032] The nanoparticles are selected from one or more of the following: colloidal gold, colloidal carbon, gold microsonic@nanomicrosphere, preferably colloidal gold or gold microsonic@nanomicrosphere, most preferably gold microsonic@nanomicrosphere;

[0033] The nanoparticle-labeled cortisol monoclonal antibody is prepared by mixing the nanoparticles and the cortisol monoclonal antibody; the concentration of the cortisol monoclonal antibody is preferably 0.1-0.5 mg / mL, more preferably 0.2-0.45 mg / mL, and further preferably 0.25-0.40 mg / mL;

[0034] The nanoparticle-labeled IgY is prepared by mixing the nanoparticles and the IgY; the concentration of the IgY is preferably 0.1-5 mg / mL, more preferably 0.5-1.5 mg / mL, and further preferably 1.0-1.2 mg / mL; and / or

[0035] The concentration of the colloidal gold in the nanoparticles is preferably 10-100 OD, more preferably 20-80 OD, and further preferably 30-60 OD; the concentration of the colloidal carbon is preferably 1x colloidal carbon-10x colloidal carbon, more preferably 3x colloidal carbon-7x colloidal carbon, and further preferably 5.5x colloidal carbon-6.5x colloidal carbon; and the concentration of the gold microsub-nanomicrosphere label is preferably 1-50 OD, more preferably 10-40 OD, and further preferably 15-25 OD.

[0036] Preferably, the volume ratio of the nanoparticles to the IgY is 50-90:1, more preferably 55-80:1, and further preferably 60-70:1.

[0037] Preferably, the volume ratio of the nanoparticles to the cortisol monoclonal antibody is 20-50:1, more preferably 30-40:1, and further preferably 35-37:1.

[0038] Preferably, the particle size of the colloidal gold is 20-80 nm, more preferably 30-70 nm, and further preferably 40-60 nm.

[0039] Preferably, the particle size of the colloidal carbon is 80-500 nm, more preferably 100-450 nm, and further preferably 150-400 nm; and / or

[0040] Preferably, the particle size of the gold microsub-nanomicrosphere label is 250-450 nm, more preferably 280-350 nm, and further preferably 300-330 nm.

[0041] The saliva cortisol rapid immunochromatographic strip according to the first aspect of the present application, wherein,

[0042] The concentration of the nanoparticle-labeled cortisol monoclonal antibody is 0.15 mg / mL-0.6 mg / mL, preferably 0.2 mg / mL-0.55 mg / mL, and more preferably 0.20 mg / mL-0.40 mg / mL.

[0043] The concentration of the nanoparticle-labeled IgY is 0.5 mg / mL-4 mg / mL, preferably 1.0 mg / mL-3.5 mg / mL, and more preferably 1.1 mg / mL-3.0 mg / mL; and / or

[0044] The volume ratio of the nanoparticle-labeled cortisol monoclonal antibody to the nanoparticle-labeled IgY is 1-6:1, preferably 1-4:1, and more preferably 1-2:1.

[0045] The saliva cortisol rapid immunochromatographic strip according to the first aspect of the present application, wherein the saliva cortisol rapid immunochromatographic strip further comprises a card shell, and wherein:

[0046] The faceplate of the card shell comprises an observation area and a sample adding area.

[0047] The saliva cortisol rapid immunochromatographic strip according to the first aspect of the present application, wherein the conjugate pad further comprises a quality control C line and a detection T line; wherein:

[0048] The quality control C line and the detection T line are located in the observation area, and the detection T line is located between the quality control C line and the sample adding area.

[0049] Preferably, the quality control C line is coated with a solution comprising anti-IgY; and / or

[0050] Preferably, the detection T line is coated with a solution comprising cortisol-coupled bovine serum albumin.

[0051] The second aspect of the present application provides a method for preparing the saliva cortisol rapid immunochromatographic strip according to the first aspect, the method comprising the following steps:

[0052] (1) respectively preparing the nanoparticle-labeled cortisol monoclonal antibody and the nanoparticle-labeled IgY;

[0053] (2) mixing the nanoparticle-labeled cortisol monoclonal antibody and the nanoparticle-labeled IgY prepared in step (1), and spraying them onto the conjugate pad;

[0054] (3) respectively preparing a quality control C line dotting membrane solution and a detection T line dotting membrane solution, and dotting membranes;

[0055] (4) sequentially lapping a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent paper on a PVC base plate, placing them into a card shell, and drying and sealing to obtain the saliva cortisol rapid immunochromatographic strip.

[0056] The method according to the second aspect of the present application, wherein in step (1):

[0057] The preparation method of the nanoparticle-labeled cortisol monoclonal antibody comprises: sequentially adding water and a pH adjuster into the nanoparticles, stirring respectively, then adding a cortisol monoclonal antibody, adding a blocking agent for blocking, centrifuging to remove supernatant, then adding a reconstitution solution, and obtaining the nanoparticle-labeled cortisol monoclonal antibody after reaction; and / or

[0058] The preparation method of the nanoparticle-labeled IgY comprises: sequentially adding water and a pH adjuster into the nanoparticles, stirring respectively, then adding IgY, adding a blocking agent for blocking, centrifuging to remove supernatant, then adding a reconstitution solution, and obtaining the nanoparticle-labeled IgY after reaction.

[0059] Preferably, the pH adjusting agent is selected from one or more of: potassium carbonate, sodium carbonate, potassium hydroxide, sodium hydroxide, sodium bicarbonate, potassium bicarbonate, more preferably from one or more of: potassium carbonate, potassium hydroxide, potassium bicarbonate, most preferably potassium carbonate;

[0060] Preferably, the blocking agent is selected from one or more of: bovine serum albumin, casein, sodium caseinate, more preferably bovine serum albumin and / or sodium caseinate; and / or

[0061] Preferably, the reconstitution solution comprises: a buffer, a stabilizer, a liquid diffusion control agent, and a preservative; wherein the preservative is preferably selected from one or more of: ProClin 300, Proclin 950, KroVin 300, KroVin 950, Rozone S16.

[0062] The method according to the second aspect of the present application, wherein the step (2) further comprises: soaking the sample pad and the conjugate pad in a treatment solution, and drying for standby, before spraying onto the conjugate pad; wherein,

[0063] The treatment solution comprises: a buffer, a blocking agent, a high molecular polymer protective agent, and a surfactant;

[0064] Preferably, the buffer is selected from one or more of: phosphate buffered saline, Dulbecco's phosphate buffered saline, citric acid-sodium citrate buffer, boric acid-borax buffer, citric acid-borax buffer, more preferably phosphate buffered saline or Dulbecco's phosphate buffered saline;

[0065] Preferably, the pH value of the buffer is 7.0-8.5, more preferably 7.0-8.0, further preferably 7.2-7.4;

[0066] Preferably, the high molecular polymer is selected from one or more of: PEG6000, PEG20000, PVP-3000, polyvinyl alcohol, polyacrylamide, polyacrylic acid, more preferably PEG6000 and / or PVP-3000;

[0067] Preferably, the protective agent is selected from one or more of: sucrose, heteropolysaccharide, chitosan, trehalose, more preferably sucrose or trehalose;

[0068] Preferably, the blocking agent is selected from one or more of: casein, sodium caseinate, bovine serum albumin, skimmed milk powder, gelatin, more preferably bovine serum albumin or sodium caseinate; and / or

[0069] Preferably, the surfactant is selected from one or more of the following: Tween-20, Tween-80, surfactant S6, surfactant S9, surfactant S17, surfactant S21, alkylphenol polyoxyethylene ether, dodecyl dimethyl betaine, fatty alcohol polyoxyethylene ether, polyoxyethylene lauryl ether; more preferably, it is selected from one or more of the following: Tween-20, Tween-80, surfactant S6, surfactant S9, surfactant S17, surfactant S21.

[0070] According to the method of the second aspect of the present invention, in step (3):

[0071] The preparation method of the quality control C-line streaking solution includes: adding streaking solution to anti-IgY; and / or

[0072] The method for preparing the T-line detection membrane solution includes: adding cortisol-conjugated bovine serum albumin to the streaking solution;

[0073] Preferably, the method for preparing the streaking solution includes: adding trehalose and / or sucrose to the buffer solution to obtain the streaking solution;

[0074] Preferably, the concentration of cortisol-conjugated bovine serum albumin is 0.1 mg / mL to 5 mg / mL, more preferably 0.5 mg / mL to 3 mg / mL, and even more preferably 1 mg / mL to 2 mg / mL;

[0075] Preferably, the concentration of the anti-IgY is 0.1 mg / mL to 5 mg / mL, more preferably 0.5 mg / mL to 3 mg / mL, and even more preferably 1 mg / mL to 2 mg / mL; and / or

[0076] Preferably, the amount of the quality control C-line spot membrane solution and the detection T-line spot membrane solution applied is 0.5–2.0 μL / cm, more preferably 1.0–1.5 μL / cm, and most preferably 1 μL / cm.

[0077] A third aspect of the invention provides the use of the rapid salivary cortisol immunochromatographic strip described in the first aspect in the preparation of salivary cortisol detection products.

[0078] The salivary cortisol rapid immunochromatographic strip of the present invention may have, but is not limited to, the following beneficial effects:

[0079] The gold probe-based rapid immunochromatographic strip for salivary cortisol of this invention is of great significance for the diagnosis and prognosis monitoring of Cushing's syndrome. It also has some indicative value for mental health disorders such as stress and depression. Attached Figure Description

[0080] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0081] Figure 1 A structural diagram of the salivary cortisol rapid immunochromatographic strip of the present invention is shown.

[0082] Figure 2 The results of test strips with different cortisol concentrations in Experiment Example 1 are shown.

[0083] Figure 3 The results of real saliva test strips from different people in Experiment Example 1 are shown.

[0084] Figure 4 The diagram shows the effect of the salivary cortisol rapid immunochromatographic strip prepared in Example 1 at different dilutions after being labeled with nanoparticle antibodies.

[0085] Figure 5 The following are illustrative images of the effects of the salivary cortisol rapid immunochromatographic strips prepared in Examples 2-4.

[0086] Figure 6 The diagram shows the effects of different amounts of nanoparticles in Experiment 1. The volumes of gold microspheres from left to right are 100 μL, 125 μL, 150 μL, 175 μL, and 200 μL.

[0087] Figure 7 The effect of different amounts of EDTA in the sample pad treatment solution in Experiment Example 1 is shown in the figure. The mass fraction of EDTA from left to right is 0%, 0.05%, 0.1%, 0.5%, and 1%.

[0088] Figure 8 The diagram shows the effects of different surfactants in the sample pad treatment solution of Experimental Example 1. From left to right, they are: Tween-20+ surfactant S6, Tween-20+ surfactant S9, Tween-20+ surfactant S21, Tween-20+ surfactant S17, and Tween 80+ surfactant S6.

[0089] Figure 9 The diagram shows the effects of different surfactants in the sample pad treatment solution of Experimental Example 1. From left to right, they are: Tween-80 + surfactant S9, Tween-80 + surfactant S21, Tween-80 + surfactant S17, surfactant S9 + surfactant S21, and surfactant S6 + surfactant S17.

[0090] Explanation of reference numerals in the attached figures:

[0091] 1. Sample pad; 2. Binding pad; 3. Nitrocellulose membrane; 4. Absorbent paper. Detailed Implementation

[0092] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for more detailed and specific illustration and should not be construed as limiting the present invention in any way.

[0093] This section provides a general description of the materials and testing methods used in the experiments of this invention. While many of the materials and methods of operation used to achieve the objectives of this invention are well known in the art, the invention is still described in as much detail as possible herein. It will be apparent to those skilled in the art that, unless otherwise stated in the context, the materials and methods of operation used in this invention are well known in the art.

[0094] The reagents and instruments used in the following examples are as follows:

[0095] Reagents:

[0096] Chicken IgY, purchased from Chongqing Wokawei Biotechnology Co., Ltd.

[0097] Binding pad: purchased from Shanghai Jiening Biotechnology Co., Ltd.

[0098] Sample pad: purchased from Hangzhou Zehuai Medical Devices Co., Ltd.

[0099] Absorbent paper, purchased from Shanghai Jinbiao Biotechnology Co., Ltd.

[0100] NC membrane and Tween-20 were purchased from Xibao Biotechnology (Shanghai) Co., Ltd.

[0101] Colloidal gold particles with a diameter of 20-310 nm were purchased from Wanhua Puman Biotechnology Co., Ltd.

[0102] Gold microspheres@nanospheres were purchased from Zhengzhou Lingsi Biotechnology Co., Ltd.

[0103] Na2HPO4·2H2O, NaH2PO4·H2O, casein, polyethylene glycol (average molecular weight 6000), trehalose, polyvinylpyrrolidone-3000, and bovine serum albumin were all purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0104] instrument:

[0105] The gold spraying apparatus was purchased from Shanghai Jinbiao Biotechnology Co., Ltd.

[0106] Example 1

[0107] This embodiment illustrates the preparation method of the salivary cortisol rapid immunochromatographic strip of the present invention.

[0108] Figure 1A structural diagram of the salivary cortisol rapid immunochromatographic strip of the present invention is shown. The salivary cortisol rapid immunochromatographic strip includes: a sample pad 1, a conjugate pad 2, a nitrocellulose membrane 3, and absorbent paper 4, which are sequentially overlapped on a PVC base plate.

[0109] Its preparation method is as follows:

[0110] 1. Prepare the following solutions:

[0111] ①Preparation method for pH 7.4, 0.01 mol / L phosphate buffered PBS:

[0112] The concentration of Na2HPO4·2H2O 0.01M solution is 1.781 g / L (No. 1);

[0113] NaH2PO4·H2O: 0.01M solution has a concentration of 1.38 g / L (No. 2);

[0114] Add 81 mL of solution 1 and 19 mL of solution 2.

[0115] ② Method for preparing the reconstitution solution:

[0116] PBS 7.4, 0.5% BSA, 0.1% PEG8000, 0.1% PEG20000, 0.05% ProClin300.

[0117] ③ Method for preparing the marking fluid:

[0118] Add 1g of trehalose to 100mL of 0.01mol / L pH 7.4 PBS.

[0119] ④ Preparation method of sample pad and conjugate pad treatment solution:

[0120] Mix 100 mL of 0.01 mol / L pH 7.4 PBS, 0.5% casein, 1% polyethylene glycol (average molecular weight 6000), 5% trehalose, 0.1% polyvinylpyrrolidone-3000, 1% bovine serum albumin, and 1% Tween-20 by sonication until homogeneous and ready for use.

[0121] 2. Preparation of gold-labeled antibodies

[0122] (1) Preparation of gold-labeled cortisol monoclonal antibody: 200 μL of gold (particle size: 40 nm, concentration: 100 OD) was added to 800 μL of water, stirred evenly, and then 8 μL of K2CO3 was added. The mixture was stirred evenly again, and then cortisol monoclonal antibody was added to make the final concentration 0.4 mg / mL. The reaction was carried out at room temperature for 1 h. 1 mL of 0.5% BSA was added for blocking, and the reaction was carried out at room temperature for 2 h. Then, the mixture was centrifuged at 12000 r / min for 10 min at 4 °C, the supernatant was removed, and 25 μL of reconstitution solution was added.

[0123] (2) Preparation of gold-labeled chicken IgY: 200 μL of gold (particle size: 40 nm, concentration: 100 OD) was added to 800 μL of water and stirred until homogeneous. Then, 5 μL of K2CO3 was added and stirred until homogeneous again. Chicken IgY was then added to bring the final concentration to 3 mg / mL, and the mixture was reacted at room temperature for 1 h. 1 mL of 0.5% BSA was added for blocking, and the mixture was reacted at room temperature for 2 h. Then, the mixture was centrifuged at 12000 r / min for 10 minutes at 4 °C, the supernatant was removed, and 25 μL of reconstitution solution was added.

[0124] (3) Sample pad and conjugate pad treatment: Immerse the conjugate pad and sample pad in the sample pad and conjugate pad treatment solution prepared in step 1 for 1 hour, and dry at 37°C for 4 hours.

[0125] (4) Spraying with gold: Combine the solutions prepared in step (1) and step (2) at a volume ratio of 1:1, mix them well, and use a gold spraying device to spray the gold-labeled corticosteroid monoclonal antibody and gold-labeled chicken IgY onto the conjugate pad at a spraying volume of 3 μL / cm.

[0126] 3. Dot film

[0127] Add 3 μL of anti-chicken IgY to 30 μL of streak solution as the quality control C line.

[0128] Add 2 μL of cortisol-BSA to 30 μL of streak buffer to form the T line.

[0129] Film application rate: 1 μL / cm

[0130] 4. Assembly: Length x Width

[0131] The prepared sample pad (2cm x 10cm), conjugate pad (0.5cm x 10cm), NC membrane (2.5cm x 10cm), and absorbent paper (2.8cm x 10cm) are overlapped by 2mm in two parts and then glued to a PVC base plate. They are then cut into 0.4cm wide test strips using a cutting machine, placed in a card holder, and sealed in an aluminum foil bag with desiccant for storage.

[0132] Examples 2 to 8

[0133] This embodiment illustrates the preparation method of the salivary cortisol rapid immunochromatographic strip of the present invention.

[0134] Except for those listed in Table 1, the preparation methods of Examples 2-8 are the same as those of Example 1.

[0135] Table 1. Salivary cortisol rapid immunochromatographic strips prepared in Examples 2-8

[0136]

[0137]

[0138] Test Example 1

[0139] This embodiment is used to illustrate the effect of the salivary cortisol rapid immunochromatographic strip of the present invention.

[0140] This embodiment uses the salivary cortisol rapid immunochromatographic strips prepared in Examples 1-8 to test for cortisol in artificial saliva. The test method is as follows: 100 μL of artificial saliva is added to the sample well of the test strip, reacted at room temperature for 10 min, and the results are observed with the naked eye. The concentrations of cortisol are 0, 1, 1.45, 2, 2.72, 3, and 4 ng / mL, respectively. Figure 2 The test results of different cortisol concentration test strips in Example 1 are shown. Figure 3 The results of real saliva test strips from different people in Experiment Example 1 are shown.

[0141] from Figure 2 As can be seen, the T-line gradually weakens with increasing cortisol concentration. The changes in the T-line are particularly pronounced at 1.45 ng / mL and 2.72 ng / mL. Note: 1.45 ng / mL is considered the critical value for midnight salivary cortisol in domestic and international expert guidelines, public knowledge, and literature; 2.72 ng / mL is the threshold value for midnight salivary cortisol post-treatment monitoring. Figure 3 These are color images of salivary cortisol test strips from different individuals. The saliva was collected using a saliva collection tube without any other pretreatment. Figure 2 and Figure 3 It can be seen that the salivary cortisol rapid immunochromatographic strips of the present invention have good detection effects.

[0142] Figure 4 The diagram shows the effect of the salivary cortisol rapid immunochromatographic strip prepared in Example 1 at different dilution ratios. Figure 4 It can be seen that the T and C lines of the salivary cortisol rapid immunochromatographic strip prepared in Example 1 showed that the nanoparticle-labeled antibody had the best effect when diluted.

[0143] like Figure 5 As shown, the salivary cortisol rapid immunochromatographic strips prepared in Examples 2-4 can all display T and C lines normally. Figure 6 The effect of different amounts of nanoparticles in Experiment Example 1 is shown in the diagram. Figure 7 The effect of different amounts of EDTA in the sample pad treatment solution in Experimental Example 1 is shown in the graph. For example... Figure 8 and 9 As shown, different surfactants have a significant impact on the test results.

[0144] Comparative Example 1

[0145] This comparative example is used to compare the salivary cortisol rapid immunochromatographic strip of the present invention with other test strips.

[0146] As shown in Table 2, compared with the prior art, the salivary cortisol rapid immunochromatographic strip of the present invention can accurately detect the level of cortisol in saliva.

[0147] Table 2 Comparison of the salivary cortisol rapid immunochromatographic strip of the present invention with other test strips

[0148]

[0149]

[0150]

[0151] While the effects of some embodiments have been shown above, those skilled in the art should understand that, based on the concept of the invention, other embodiments not specifically shown or other technical solutions of the invention not shown in the embodiments can also achieve the same technical effects as those claimed in the summary section:

[0152] The present invention relates to a rapid salivary cortisol immunochromatographic strip based on a colloidal gold probe, which is of great significance for the diagnosis and prognosis monitoring of Cushing's syndrome. It also has certain indicative significance for mental health disorders such as stress and depression.

[0153] Although the invention has been described to a certain extent, it is apparent that appropriate variations can be made to the various conditions without departing from the spirit and scope of the invention. It is understood that the invention is not limited to the described embodiments, but falls within the scope of the claims, which include equivalent substitutions for each of the elements.

Claims

1. A salivary cortisol rapid immunochromatographic strip, characterized by, The saliva cortisol rapid immunochromatography strip comprises, in sequence, a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent paper on a PVC base plate. The conjugate pad is sprayed with nano-particle labeled cortisol monoclonal antibody and nano-particle labeled IgY, preferably selected from one or more of the following: avian IgY, reptile IgY, amphibian IgY, preferably selected from one or more of the following: chicken IgY, duck IgY, goose IgY, most preferably chicken IgY.

2. The saliva cortisol rapid immunochromatography strip according to claim 1, characterized in that: The nano-particles are selected from one or more of the following: colloidal gold, colloidal carbon, gold micro-son@nanomicrosphere, preferably colloidal gold or gold micro-son@nanomicrosphere, most preferably gold micro-son@nanomicrosphere; The nano-particle labeled cortisol monoclonal antibody is prepared by mixing the nano-particles and cortisol monoclonal antibody; the concentration of the cortisol monoclonal antibody is preferably 0.1-0.5 mg / mL, more preferably 0.2-0.45 mg / mL, and further preferably 0.25-0.40 mg / mL; The nano-particle labeled IgY is prepared by mixing the nano-particles and IgY; the concentration of the IgY is preferably 0.1-5 mg / mL, more preferably 0.5-1.5 mg / mL, and further preferably 1.0-1.2 mg / mL; and / or The concentration of the colloidal gold in the nano-particles is preferably 10-100 OD, more preferably 20-80 OD, and further preferably 30-60 OD; the concentration of the colloidal carbon is preferably 1x colloidal carbon-10x colloidal carbon, more preferably 3x colloidal carbon-7x colloidal carbon, and further preferably 5.5x colloidal carbon-6.5x colloidal carbon; and the concentration of the gold micro-son@nanomicrosphere label is preferably 1-50 OD, more preferably 10-40 OD, and further preferably 15-25 OD; Preferably, the volume ratio of the nano-particles to the IgY is 50-90:1, more preferably 55-80:1, and further preferably 60-70:1; Preferably, the volume ratio of the nano-particles to the cortisol monoclonal antibody is 20-50:1, more preferably 30-40:1, and further preferably 35-37:1; Preferably, the particle size of the colloidal gold is 20-80 nm, more preferably 30-70 nm, and further preferably 40-60 nm; Preferably, the particle size of the colloidal carbon is 80-500 nm, more preferably 100-450 nm, and further preferably 150-400 nm; and / or Preferably, the particle size of the gold micro-son@nanomicrosphere label is 250-450 nm, more preferably 280-350 nm, and further preferably 300-330 nm.

3. The saliva cortisol rapid immunochromatography strip according to claim 1 or 2, characterized in that: The concentration of the nanoparticle-labeled cortisol monoclonal antibody is 0.15 mg / mL to 0.6 mg / mL, preferably 0.2 mg / mL to 0.55 mg / mL, and more preferably 0.20 mg / mL to 0.40 mg / mL; The concentration of the nanoparticle-labeled IgY is 0.5 mg / mL to 4 mg / mL, preferably 1.0 mg / mL to 3.5 mg / mL, and more preferably 1.1 mg / mL to 3.0 mg / mL; and / or The volume ratio of the nanoparticle-labeled cortisol monoclonal antibody to the nanoparticle-labeled IgY is 1 to 6:1, preferably 1 to 4:1, and more preferably 1 to 2:

1.

4. The salivary cortisol rapid immunochromatographic strip according to any one of claims 1 to 3, characterized in that, The saliva cortisol rapid immunochromatographic strip further comprises a card shell, wherein: The panel of the card shell comprises an observation area and a sample addition area.

5. The salivary cortisol rapid immunochromatographic strip according to claim 4, characterized by, The binding pad further comprises a quality control C line and a detection T line, wherein: The quality control C line and the detection T line are located in the observation area, and the detection T line is located between the quality control C line and the sample addition area; Preferably, the quality control C line is coated with a solution containing anti-IgY; and / or Preferably, the detection T line is coated with a solution containing cortisol-coupled bovine serum albumin.

6. A method for the preparation of a salivary cortisol rapid immunochromatographic strip according to any one of claims 1 to 5, characterized in that, The method comprises the following steps: (1) separately preparing the nanoparticle-labeled cortisol monoclonal antibody and the nanoparticle-labeled IgY; (2) mixing the nanoparticle-labeled cortisol monoclonal antibody and the nanoparticle-labeled IgY prepared in step (1), and spraying them onto the binding pad; (3) separately preparing a quality control C line point membrane solution and a detection T line point membrane solution, and performing point membrane; (4) sequentially laminating a sample pad, a binding pad, a nitrocellulose membrane, and an absorbent paper on a PVC base plate, placing them into a card shell, and drying and sealing to obtain the saliva cortisol rapid immunochromatographic strip.

7. The method of claim 6, wherein, In step (1), the preparation method of the nanoparticle-labeled cortisol monoclonal antibody comprises: sequentially adding water and a pH adjuster into the nanoparticles, stirring separately, then adding cortisol monoclonal antibody, adding a blocking agent for blocking, centrifuging to remove the supernatant, then adding a reconstitution solution, and obtaining the nanoparticle-labeled cortisol monoclonal antibody after reaction; and / or The preparation method of the nanoparticle-labeled IgY comprises: sequentially adding water and a pH adjuster into the nanoparticles, stirring separately, then adding IgY, adding a blocking agent for blocking, centrifuging to remove the supernatant, then adding a reconstitution solution, and obtaining the nanoparticle-labeled IgY after reaction; Preferably, the pH adjuster is selected from one or more of the following: potassium carbonate, sodium carbonate, potassium hydroxide, sodium hydroxide, sodium bicarbonate, and potassium bicarbonate, more preferably selected from one or more of the following: potassium carbonate, potassium hydroxide, and potassium bicarbonate, and most preferably potassium carbonate; Preferably, the blocking agent is selected from one or more of the following: bovine serum albumin, casein, and sodium caseinate, more preferably bovine serum albumin and / or sodium caseinate; and / or Preferably, the blocking agent is selected from one or more of the following: bovine serum albumin, casein, and sodium caseinate, more preferably bovine serum albumin and / or sodium caseinate; and / or Preferably, the reconstitution solution comprises: a buffer, a stabilizer, a liquid diffusion control agent, and a preservative; wherein the preservative is preferably selected from one or more of the following: ProClin 300, Proclin 950, KroVin 300, KroVin 950, Rozone S16.

8. The method according to claim 6 or 7, characterized in that, In the step (2), before being sprayed onto the conjugate pad, the sample pad and the conjugate pad are further soaked in a treatment solution and dried for standby use; wherein, The treatment solution comprises: a buffer, a blocking agent, a high-molecular polymer protective agent, and a surfactant; Preferably, the buffer is selected from one or more of the following: phosphate buffered saline, Dulbecco's phosphate buffered saline, citric acid-sodium citrate buffer, boric acid-borax buffer, citric acid-borax buffer, more preferably phosphate buffered saline or Dulbecco's phosphate buffered saline; Preferably, the pH value of the buffer is 7.0-8.5, more preferably 7.0-8.0, and further preferably 7.2-7.4; Preferably, the high-molecular polymer is selected from one or more of the following: PEG6000, PEG20000, PVP-3000, polyvinyl alcohol, polyacrylamide, polyacrylic acid, and more preferably PEG6000 and / or PVP-3000; Preferably, the protective agent is selected from one or more of the following: sucrose, heteropolysaccharide, chitosan, trehalose, and more preferably sucrose or trehalose; Preferably, the blocking agent is selected from one or more of the following: casein, sodium caseinate, bovine serum albumin, skimmed milk powder, gelatin, and more preferably bovine serum albumin or sodium caseinate; and / or Preferably, the surfactant is selected from one or more of the following: Tween-20, Tween-80, surfactant S6, surfactant S9, surfactant S17, surfactant S21, alkylphenol polyoxyethylene ether, dodecyl dimethyl betaine, fatty alcohol polyoxyethylene ether, polyoxyethylene lauryl ether, and more preferably selected from one or more of the following: Tween-20, Tween-80, surfactant S6, surfactant S9, surfactant S17, surfactant S21.

9. The method according to any one of claims 6 to 8, characterized in that, In the step (3): The preparation method of the quality control C-line dot membrane solution comprises: adding a marking solution to anti-IgY; and / or The preparation method of the detection T-line dot membrane solution comprises: adding cortisol-coupled bovine serum albumin to the marking solution; Preferably, the preparation method of the marking solution comprises: adding trehalose and / or sucrose to the buffer to obtain the marking solution; Preferably, the concentration of the cortisol-coupled bovine serum albumin is 0.1 mg / mL-5 mg / mL, more preferably 0.5 mg / mL-3 mg / mL, and further preferably 1 mg / mL-2 mg / mL; Preferably, the concentration of the anti-IgY is 0.1 mg / mL-5 mg / mL, more preferably 0.5 mg / mL-3 mg / mL, and further preferably 1 mg / mL-2 mg / mL; and / or Preferably, the concentration of the anti-IgY is 0.1 mg / mL-5 mg / mL, more preferably 0.5 mg / mL-3 mg / mL, and further preferably 1 mg / mL-2 mg / mL; and / or Preferably, the amount of the quality control C-line spotting solution and the amount of the detection T-line spotting solution are 0.5-2.0 μL / cm, preferably 1.0-1.5 μL / cm, and most preferably 1 μL / cm.

10. Use of the salivary cortisol rapid immunochromatographic strip according to any one of claims 1 to 5 in the preparation of a product for the detection of salivary cortisol.

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