Test strip for simultaneously detecting five oral tumor markers, preparation method and kit

Abstract

The invention belongs to the technical field of in-vitro diagnosis, and relates to a test strip for simultaneously detecting five oral tumor markers, a preparation method and a kit, the test strip comprises a sample pad, a combination pad, a reaction film and an absorption pad which are sequentially overlapped on a bottom plate; the combination pad contains an animal anti-human CEA monoclonal antibody, an animal anti-human SCC monoclonal antibody, an animal anti-human LAP monoclonal antibody, an animal anti-human LSA monoclonal antibody and an animal anti-human GST monoclonal antibody which are respectively marked by colloidal gold; a first detection line coated with a CEA antibody, a second detection line coated with an SCC antibody, a third detection line coated with an LAP antibody, a fourth detection line coated with an LSA antibody, a fifth detection line coated with a GST antibody and a quality control line coated with an animal IgG antibody are arranged on the reaction film. According to the invention, five different markers of oral tumors can be detected at the same time, the operation is simple, and the detection time is short; the colloidal gold technology is adopted, the detection sensitivity is high, the specificity is good, and the result is accurate.

Description

Technical Field

[0001] The present application relates to the field of in vitro diagnostic technology, and in particular to a test strip for simultaneously detecting five oral tumor markers, a preparation method and a kit. Background Art

[0002] Common oral tumor markers include carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCCA), immunosuppressive acidic protein (LAP), lipid-bound sialic acid (LSA), glutathione S-transferase (GST) and other substances. Studies have shown that in oral squamous cell carcinoma, the rate of CEA is high, and the positive rate is significantly higher than that of normal people; SCC-Ag in serum, plasma, and whole blood of patients with oral squamous cell carcinoma increases. After the patient's tumor relapses, the positive rate increases to 75%, and its level decreases significantly after treatment, which has special significance for the efficacy detection of patients with oral squamous cell carcinoma; cancer cells have the ability to produce and secrete LAP. The serum, plasma, and whole blood LAP content of patients with malignant tumors is significantly higher than that of patients with both sexes and normal people; LSA is sialic acid bound to gangliosides, which is closely related to the growth and decline, recurrence, and metastasis of tumors. It has a high specificity for tumor diagnosis. In patients with oral cancer, it reflects the tumor load and has a good correlation with the tumor. It is of great significance for predicting the early malignant transformation of oral squamous cell carcinoma; in patients with different degrees of oral leukoplakia and squamous cell carcinoma, GST can be used as a marker for oral precancerous lesions and oral cancer as the degree of leukoplakia increases. The detection of the above five oral tumor markers has a certain diagnostic value for oral tumors.

[0003] At present, the commonly used detection methods for tumor markers include radioimmunoassay and chemiluminescence, which are all based on the detection of antigen-antibody specific binding reactions. However, the reagents used in radioimmunoassay have a short half-life and high cost, and require special equipment such as scintillation counters. At the same time, radionuclides have certain potential hazards to the human body, and experimental waste disposal is difficult. Radionuclide labeling sometimes changes the physiological activity of certain biological substances; chemiluminescence requires a professional chemiluminescence instrument, and most of them are closed systems. Reagents and instruments from different manufacturers are not universal, and the cost is high. Summary of the invention

[0004] The purpose of this application is to propose a test strip, preparation method and kit for simultaneously detecting five oral tumor markers, so as to solve the technical problems of high cost and complex operation of tumor marker detection in the prior art.

[0005] In order to solve the above technical problems, the present application embodiment provides a test strip for simultaneously detecting five oral tumor markers, which adopts the following technical solution:

[0006] It includes a sample pad, a binding pad, a reaction membrane and an absorption pad, wherein the sample pad, the binding pad, the reaction membrane and the absorption pad are overlapped on a bottom plate in sequence;

[0007] The conjugate pad contains colloidal gold-labeled animal anti-human CEA monoclonal antibody, colloidal gold-labeled animal anti-human SCC monoclonal antibody, colloidal gold-labeled animal anti-human LAP monoclonal antibody, colloidal gold-labeled animal anti-human LSA monoclonal antibody and colloidal gold-labeled animal anti-human GST monoclonal antibody; the reaction membrane is provided with a first detection line coated with CEA monoclonal antibody, a second detection line coated with SCC monoclonal antibody, a third detection line coated with LAP monoclonal antibody, a fourth detection line coated with LSA monoclonal antibody, a fifth detection line coated with GST monoclonal antibody and a quality control line coated with animal IgG antibody.

[0008] Further, the test strip includes a first colloidal gold test paper unit and a second colloidal gold test paper unit, the sample pad includes a first sample pad and a second sample pad, the binding pad includes a first binding pad and a second binding pad, the reaction membrane includes a first reaction membrane and a second reaction membrane, and the absorption pad includes a first absorption pad and a second absorption pad;

[0009] The first colloidal gold test paper unit is composed of the first sample pad, the first conjugation pad, the first reaction membrane and the first absorption pad which are sequentially overlapped on the bottom plate; the second colloidal gold test paper unit is composed of the second sample pad, the second conjugation pad, the second reaction membrane and the second absorption pad which are sequentially overlapped on the bottom plate;

[0010] The first conjugate pad contains colloidal gold-labeled animal anti-human CEA monoclonal antibodies and colloidal gold-labeled animal anti-human SCC monoclonal antibodies; the second conjugate pad contains colloidal gold-labeled animal anti-human LAP monoclonal antibodies, colloidal gold-labeled animal anti-human LSA monoclonal antibodies and colloidal gold-labeled animal anti-human GST monoclonal antibodies; the first reaction membrane is provided with a first detection line coated with CEA monoclonal antibodies, a second detection line coated with SCC monoclonal antibodies and a quality control line coated with animal IgG antibodies; the second reaction membrane is provided with a third detection line coated with LAP monoclonal antibodies, a fourth detection line coated with LSA monoclonal antibodies, a fifth detection line coated with GST monoclonal antibodies and a quality control line coated with animal IgG antibodies.

[0011] Furthermore, the colloidal gold is selected from nano-gold particles, and the diameter of the nano-gold particles is 90-110nm; the species of the animals include rabbits, sheep, chickens and mice.

[0012] In order to solve the above technical problems, the present application also provides a method for preparing a test strip for simultaneously detecting five oral tumor markers, which adopts the following technical solution:

[0013] Prepare sample pads, conjugate pads, reaction membranes and absorption pads;

[0014] Cover one end of the sample pad with one end of the conjugate pad, cover the other end of the conjugate pad with the end of the reaction membrane away from the quality control line, cover one end of the absorption pad with the other end of the reaction membrane close to the quality control line, and stick them on the bottom plate in sequence to form a test strip;

[0015] Wherein, the conjugate pad is prepared by the following preparation steps:

[0016] Prepare colloidal gold solution;

[0017] Using the colloidal gold solution to label animal anti-human CEA monoclonal antibody, animal anti-human SCC monoclonal antibody, animal anti-human LAP monoclonal antibody, animal anti-human LSA monoclonal antibody and animal anti-human GST monoclonal antibody, respectively, to obtain colloidal gold labeled CEA antibody solution, colloidal gold labeled SCC antibody solution, colloidal gold labeled LAP antibody solution, colloidal gold labeled LSA antibody solution and colloidal gold labeled GST antibody solution;

[0018] The colloidal gold labeled CEA antibody solution, the colloidal gold labeled SCC antibody solution, the colloidal gold labeled LAP antibody solution, the colloidal gold labeled LSA antibody solution and the colloidal gold labeled GST antibody solution are mixed according to a preset volume ratio to obtain a labeled mixture;

[0019] spraying the marking mixture onto glass fiber to obtain the conjugate pad; and

[0020] The reaction membrane is prepared by the following preparation steps:

[0021] Prepare a first coating working solution containing CEA monoclonal antibody, a second coating working solution containing SCC monoclonal antibody, a third coating working solution containing LAP monoclonal antibody, a fourth coating working solution containing LSA monoclonal antibody, and a fifth coating working solution containing GST monoclonal antibody;

[0022] The first coating working solution, the second coating working solution, the third coating working solution, the fourth coating working solution and the fifth coating working solution are respectively streaked on the nitrocellulose membrane to form a first detection line, a second detection line, a third detection line, a fourth detection line and a fifth detection line;

[0023] Prepare the quality control line coating working solution containing animal IgG antibodies;

[0024] Streaking the quality control line coating working solution onto a nitrocellulose membrane to form a quality control line;

[0025] The nitrocellulose membrane after the filming is dried to obtain a reaction membrane.

[0026] Furthermore, the sample pad is prepared by the following preparation steps:

[0027] Prepare sample pad treatment solution;

[0028] The glass fiber is immersed in the sample pad treatment solution and dried to obtain the sample pad.

[0029] Furthermore, the colloidal gold solution is prepared by the following preparation steps:

[0030] Put double distilled water into a beaker and heat to boiling;

[0031] Add gold chloride aqueous solution and continue heating until boiling;

[0032] Rapidly add trisodium citrate solution to the boiling gold chloride aqueous solution, continue heating and boiling until the color of the solution changes from black to wine red, stop heating, and obtain a colloidal gold solution;

[0033] The colloidal gold solution was naturally cooled to room temperature, fixed to volume with purified water, and stored at room temperature for later use.

[0034] Further, the steps of using the colloidal gold solution to label animal anti-human CEA monoclonal antibodies, animal anti-human SCC monoclonal antibodies, animal anti-human LAP monoclonal antibodies, animal anti-human LSA monoclonal antibodies and animal anti-human GST monoclonal antibodies respectively to obtain colloidal gold labeled CEA antibody solution, colloidal gold labeled SCC antibody solution, colloidal gold labeled LAP antibody solution, colloidal gold labeled LSA antibody solution and colloidal gold labeled GST antibody solution include:

[0035] Take five centrifuge tubes, add colloidal gold solution to each tube, and add potassium carbonate solution to each tube to adjust the pH value;

[0036] Adding animal anti-human CEA monoclonal antibody, animal anti-human SCC monoclonal antibody, animal anti-human LAP monoclonal antibody, animal anti-human LSA monoclonal antibody and animal anti-human GST monoclonal antibody into different centrifuge tubes, mixing them evenly, and obtaining colloidal gold CEA antibody solution, colloidal gold SCC antibody solution, colloidal gold LAP antibody solution, colloidal gold LSA antibody solution and colloidal gold GST antibody solution respectively;

[0037] Add the labeled blocking solution to each centrifuge tube for blocking;

[0038] After the blocking reaction is completed, each centrifuge tube is centrifuged;

[0039] After centrifugation, the supernatant was discarded, and the precipitate was retained and added to the reconstitution solution to obtain colloidal gold labeled CEA antibody solution, colloidal gold labeled SCC antibody solution, colloidal gold labeled LAP antibody solution, colloidal gold labeled LSA antibody solution and colloidal gold labeled GST antibody solution.

[0040] Furthermore, the labeling blocking solution includes bovine serum albumin;

[0041] The reconstitution solution includes bovine serum albumin, sucrose, casein sodium salt, trehalose and 0.05M pH 8.0 Tris buffer.

[0042] Furthermore, the preset volume ratio is 1:1:1:1:1;

[0043] The concentrations of the first coating working solution, the second coating working solution, the third coating working solution, the fourth coating working solution and the fifth coating working solution are 1.0-1.5 mg / mL respectively;

[0044] The concentration of the quality control line coating working solution is 1.0-1.5 mg / mL.

[0045] In order to solve the above technical problems, an embodiment of the present application also provides a kit for simultaneously detecting five oral tumor markers, including a detection card, a chip and a sample diluent, characterized in that the detection card includes a card shell and a test strip prepared by the preparation method as described above; the test strip is installed in the card shell; and the chip contains standard curve data.

[0046] Compared with the prior art, this application has the following beneficial effects:

[0047] The test strip for simultaneously detecting five oral tumor markers provided in the present application can simultaneously detect five different markers of oral tumors, can comprehensively monitor oral tumors and prognosis, and make early detection of oral tumors faster and more accurate; the present application is simple to operate and has a short detection time; it adopts colloidal gold technology, has high detection sensitivity, good specificity, and accurate results, and can better meet clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the scheme in the present application, a brief introduction is given below to the drawings required for use in the description of the embodiments of the present application. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0049] Figure 1 This is a schematic diagram of the structure of a test strip for simultaneously detecting five oral tumor markers of the present application;

[0050] Figure 2 This is a schematic diagram of the structure of the first colloidal gold test paper unit of a test strip for simultaneously detecting five oral tumor markers of the present application;

[0051] Figure 3 This is a schematic diagram of the structure of the second colloidal gold test paper unit of a test strip for simultaneously detecting five oral tumor markers of the present application;

[0052] Figure 4 It is a schematic diagram of the structure of a detection card for simultaneously detecting five oral tumor markers of the present application. DETAILED DESCRIPTION

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by technicians in the technical field of the present application; the terms used in the specification of the application herein are only for the purpose of describing specific embodiments and are not intended to limit the present application; the terms "including" and "having" and any variations thereof in the specification and claims of the present application and the above-mentioned drawings are intended to cover non-exclusive inclusions. The terms "first", "second", etc. in the specification and claims of the present application or the above-mentioned drawings are used to distinguish different objects, not to describe a specific order.

[0054] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0055] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings.

[0056] This application provides a test strip for simultaneously detecting five oral tumor markers, see Figure 1 As shown, it includes a sample pad 2, a conjugation pad 3, a reaction membrane 4 and an absorption pad 5, and the sample pad 2, the conjugation pad 3, the reaction membrane 4 and the absorption pad 5 are overlapped on the bottom plate 1 in sequence.

[0057] The overlapping means that the head and tail parts of the two connected components overlap, and a connection structure that allows the liquid sample to move is formed at the overlapping part.

[0058] The conjugate pad 3 contains colloidal gold-labeled animal anti-human CEA monoclonal antibody, colloidal gold-labeled animal anti-human SCC monoclonal antibody, colloidal gold-labeled animal anti-human LAP monoclonal antibody, colloidal gold-labeled animal anti-human LSA monoclonal antibody and colloidal gold-labeled animal anti-human GST monoclonal antibody; the reaction membrane 4 is provided with a first detection line T1 coated with CEA monoclonal antibody, a second detection line T2 coated with SCC monoclonal antibody, a third detection line T3 coated with LAP monoclonal antibody, a fourth detection line T4 coated with LSA monoclonal antibody, a fifth detection line T5 coated with GST monoclonal antibody and a quality control line C coated with animal IgG antibody.

[0059] In this embodiment, the base plate may be a PVC base plate, cardboard or hardboard.

[0060] The detection principle of the test strip in this embodiment is as follows:

[0061] When the sample to be tested is added to the sample pad 2 at one end of the test strip, it moves forward by capillary action, dissolves the colloidal gold-labeled antibody on the binding pad 3 and reacts with each other, and then moves to the detection area corresponding to the reaction membrane 4. The conjugate of the sample to be tested and the colloidal gold-labeled antibody specifically binds to the corresponding antibody on the detection line and is retained, and accumulates on the detection line. The color development result can be observed by the naked eye.

[0062] In some embodiments, see Figure 2 and Figure 3 As shown, the test strip includes a first colloidal gold test paper unit and a second colloidal gold test paper unit, the sample pad 2 includes a first sample pad 21 and a second sample pad 22, the conjugate pad 3 includes a first conjugate pad 31 and a second conjugate pad 32, the reaction membrane 4 includes a first reaction membrane 41 and a second reaction membrane 42, and the absorption pad 5 includes a first absorption pad 51 and a second absorption pad 52.

[0063] The first colloidal gold test paper unit is composed of a first sample pad 21, a first conjugation pad 31, a first reaction membrane 41 and a first absorption pad 51 which are sequentially overlapped on the bottom plate 1; the second colloidal gold test paper unit is composed of a second sample pad 22, a second conjugation pad 32, a second reaction membrane 42 and a second absorption pad 52 which are sequentially overlapped on the bottom plate 1.

[0064] The first conjugation pad 31 contains colloidal gold-labeled animal anti-human CEA monoclonal antibody and colloidal gold-labeled animal anti-human SCC monoclonal antibody; the second conjugation pad 32 contains colloidal gold-labeled animal anti-human LAP monoclonal antibody, colloidal gold-labeled animal anti-human LSA monoclonal antibody and colloidal gold-labeled animal anti-human GST monoclonal antibody.

[0065] The first reaction membrane 41 is provided with a first detection line T1 coated with CEA monoclonal antibody, a second detection line T2 coated with SCC monoclonal antibody, and a quality control line C coated with animal IgG antibody; the second reaction membrane 42 is provided with a third detection line T3 coated with LAP monoclonal antibody, a fourth detection line T4 coated with LSA monoclonal antibody, a fifth detection line T5 coated with GST monoclonal antibody, and a quality control line C coated with animal IgG antibody.

[0066] It should be noted that there is no restriction on the colloidal gold labeled antibody contained on the conjugate pad of each colloidal gold test paper unit. Accordingly, there is no restriction on the antibody coated on the detection line on the reaction membrane of each colloidal gold test paper unit, as long as it matches the colloidal gold labeled antibody on the conjugate pad.

[0067] In this embodiment, the test strip is composed of two colloidal gold test paper units, which can shorten the detection time and improve the detection efficiency. At the same time, it can also avoid the long route of the sample to be tested from the sample pad to the quality control line, which may be unable to reach the line, thereby improving the accuracy of the detection.

[0068] In some embodiments, the colloidal gold is selected from nano-gold particles, and the diameter of the nano-gold particles is 90-110 nm.

[0069] Colloidal gold is formed by the polymerization of chloroauric acid (HAuCl4) into gold particles of a certain size under the action of reducing agents such as white phosphorus, ascorbic acid, sodium citrate, etc., forming a negatively charged hydrophobic colloidal solution, which condenses into a stable colloidal state due to electrostatic action, so it is usually called colloidal gold. Colloidal gold carries a negative charge in a weak alkaline environment and can form a strong bond with the positively charged groups of protein molecules. Since this bond is an electrostatic bond, it does not affect the biological properties of the protein. Therefore, colloidal gold is used as a marker to label antibodies.

[0070] When antibodies labeled with colloidal gold react with antigens, these markers aggregate on the solid phase carrier to a certain density and appear red to purple, which is visible to the naked eye. Oral tumor markers can be detected based on the color.

[0071] In this embodiment, the animal species of the antibodies include rabbit, sheep, chicken and mouse.

[0072] Among them, the animal anti-human CEA monoclonal antibody can be mouse anti-human CEA monoclonal antibody, sheep anti-human CEA monoclonal antibody or rabbit anti-human CEA monoclonal antibody, and the same applies to other antibodies.

[0073] Preferred are mouse anti-human CEA monoclonal antibody, mouse anti-human SCC monoclonal antibody, mouse anti-human LAP monoclonal antibody, mouse anti-human LSA monoclonal antibody and mouse anti-human GST monoclonal antibody.

[0074] Among them, the animal IgG antibody coated on the quality control line can be goat anti-mouse IgG antibody, goat anti-rabbit IgG antibody, etc., preferably goat anti-mouse IgG antibody.

[0075] The test strip of the present application is easy to operate and has a short detection time. Only one or two sample additions are required to obtain five test results. It can simultaneously detect five different markers of oral tumors, and can comprehensively monitor oral tumors and prognosis, making early detection of oral tumors faster and more accurate. In addition, the use of colloidal gold labeling technology makes the test strip have high detection sensitivity, good specificity, and accurate results, which better meets clinical applications.

[0076] Based on the above-mentioned test strip for simultaneously detecting five oral tumor markers, the present application also provides a test strip for simultaneously detecting five oral tumor markers, comprising the following steps:

[0077] Prepare a sample pad 2, a conjugate pad 3, a reaction membrane 4 and an absorption pad 5;

[0078] Cover one end of the sample pad 2 on one end of the conjugate pad 3, cover the other end of the conjugate pad 3 on the end of the reaction membrane 4 away from the quality control line C, cover one end of the absorption pad 5 on the other end of the reaction membrane 4 close to the quality control line C, and stick them on the bottom plate 1 in sequence to form a test strip.

[0079] The bonding pad 3 is prepared by the following steps:

[0080] Step S101, preparing a colloidal gold solution.

[0081] Specifically, double distilled water is put into a beaker and heated to boiling; an aqueous gold chloride solution is added and the solution is continuously heated to boiling; a trisodium citrate solution is rapidly added to the boiling aqueous gold chloride solution and the solution is continuously heated and boiled until the color of the solution changes from black to wine red, and the heating is stopped to obtain a colloidal gold solution; the colloidal gold solution is naturally cooled to room temperature, and the solution is made up to volume with purified water and stored at room temperature for later use.

[0082] In the present embodiment, colloidal gold nanoparticles were prepared by sodium citrate reduction method. For example, 500mL of double distilled water (ddH2O) was taken and placed in a 2000mL beaker, placed on an electric furnace and heated to boiling, 60mL of gold chloride aqueous solution with a mass concentration of 1% was added, and continued to be heated to boiling, and then about 35mL of trisodium citrate solution with a mass concentration of 2% was quickly added thereto, and continued to heat and boil for 5min, and the color of the colloidal gold solution was seen to change from black to blue-purple to wine red, and heating was stopped to obtain a colloidal gold solution. When the prepared colloidal gold solution was naturally cooled to room temperature, it was fixed to 1500mL with purified water, that is, a colloidal gold solution of one ten-thousandth with a particle size of 90nm-110nm was obtained, and stored at room temperature for standby use.

[0083] Step S102, using colloidal gold solution to label animal anti-human CEA monoclonal antibody, animal anti-human SCC monoclonal antibody, animal anti-human LAP monoclonal antibody, animal anti-human LSA monoclonal antibody and animal anti-human GST monoclonal antibody respectively, to obtain colloidal gold labeled CEA antibody solution, colloidal gold labeled SCC antibody solution, colloidal gold labeled LAP antibody solution, colloidal gold labeled LSA antibody solution and colloidal gold labeled GST antibody solution.

[0084] Taking 1 mL of colloidal gold solution as an example, the colloidal gold labeling of antibodies includes the following steps:

[0085] Step S201, take five centrifuge tubes, add colloidal gold solutions respectively, and add potassium carbonate solutions into the colloidal gold solutions to adjust the pH value.

[0086] Take five clean 2mL centrifuge tubes and use a pipette to draw 1mL of the prepared colloidal gold solution respectively; add 5-10μL of 0.2M K2CO3 solution, adjust the pH of the colloidal gold solution, and mix well.

[0087] Preparation of 0.2M K2CO3 solution: Dissolve 3.48g potassium carbonate in 80mL purified water and adjust the volume to 100mL after dissolution.

[0088] Step S202, add animal anti-human CEA monoclonal antibody, animal anti-human SCC monoclonal antibody, animal anti-human LAP monoclonal antibody, animal anti-human LSA monoclonal antibody and animal anti-human GST monoclonal antibody into different centrifuge tubes and mix them evenly to obtain colloidal gold CEA antibody solution, colloidal gold SCC antibody solution, colloidal gold LAP antibody solution, colloidal gold LSA antibody solution and colloidal gold GST antibody solution respectively.

[0089] 10 μg of mouse anti-human CEA monoclonal antibody, mouse anti-human SCC monoclonal antibody, mouse anti-human LAP monoclonal antibody, mouse anti-human LSA monoclonal antibody, and mouse anti-human GST monoclonal antibody were added to different centrifuges, and mixed evenly with the colloidal gold solution in the tube for 20 minutes to obtain the corresponding colloidal gold antibody solution.

[0090] Step S203, adding a labeled blocking solution to each centrifuge tube for blocking.

[0091] After mixing, add 100 μL of 10% labeled blocking solution to each tube for blocking and mix for 20 minutes.

[0092] Step S204: After the blocking reaction is completed, each centrifuge tube is centrifuged.

[0093] After the reaction was completed, centrifugation was performed at 4°C; the centrifugal speed was 10,000 rpm and the centrifugal time was 10 min.

[0094] Step S205, after the centrifugation is completed, the supernatant is discarded, the precipitate is retained, and the reconstitution solution is added respectively to obtain a colloidal gold labeled CEA antibody solution, a colloidal gold labeled SCC antibody solution, a colloidal gold labeled LAP antibody solution, a colloidal gold labeled LSA antibody solution and a colloidal gold labeled GST antibody solution.

[0095] After centrifugation, gently remove the centrifuge tube and slowly aspirate the supernatant using a pipette; retain the precipitate and add 100 μL of the reconstitution solution to each centrifuge tube using a pipette.

[0096] Among them, the precipitate in each centrifuge tube is the antibody gold label.

[0097] Step S103 , the colloidal gold labeled CEA antibody solution, the colloidal gold labeled SCC antibody solution, the colloidal gold labeled LAP antibody solution, the colloidal gold labeled LSA antibody solution and the colloidal gold labeled GST antibody solution are mixed according to a preset volume ratio to obtain a labeled mixture.

[0098] The preset volume ratio may be 1:1:1:1:1. That is, the colloidal gold labeled CEA antibody solution, the colloidal gold labeled SCC antibody solution, the colloidal gold labeled LAP antibody solution, the colloidal gold labeled LSA antibody solution and the colloidal gold labeled GST antibody solution are mixed at a volume ratio of 1:1:1:1:1:1.

[0099] In some embodiments, a colloidal gold-labeled CEA antibody solution and a colloidal gold-labeled SCC antibody solution are mixed in a volume ratio of 1:1:1:1:1 to obtain a labeled mixture 1; a colloidal gold-labeled LAP antibody solution, a colloidal gold-labeled LSA antibody solution, and a colloidal gold-labeled GST antibody solution are mixed in a volume ratio of 1:1:1:1:1 to obtain a labeled mixture 2.

[0100] Step S104 , spraying the marking mixture onto the glass fiber to obtain a bonding pad 3 .

[0101] Specifically, the glass fiber 8964 was cut into (10±0.5) mm×(300±10) mm, the labeling mixture was loaded into a gold spraying and scribing machine, and it was sprayed onto the cut glass fiber at a spray volume of 3.0-5.0 μL / cm to obtain a conjugate pad 3.

[0102] In some embodiments, the labeling mixture 1 and the labeling mixture 2 are loaded into a gold spraying and scribing machine and sprayed onto different cut glass fibers at a spray volume of 3.0-5.0 μL / cm to obtain a first conjugation pad 31 and a second conjugation pad 32 .

[0103] In this embodiment, the sprayed bonding pad is placed in an environment of (37±5)°C and humidity ≤30% to dry overnight (16-24) hours, and after drying, is cut into pieces with a width of 9.5-10.5 mm and a length of 290-310 mm for later use.

[0104] In this embodiment, the reaction film 4 is prepared by the following preparation steps:

[0105] Step S301, preparing a first coating working solution containing CEA monoclonal antibody, a second coating working solution containing SCC monoclonal antibody, a third coating working solution containing LAP monoclonal antibody, a fourth coating working solution containing LSA monoclonal antibody, and a fifth coating working solution containing GST monoclonal antibody.

[0106] In this embodiment, antibody coating diluents were used to dilute CEA monoclonal antibody, SCC monoclonal antibody, LAP monoclonal antibody, LSA monoclonal antibody and GST monoclonal antibody, and all were diluted to 1.0-1.5 mg / mL to prepare corresponding coating working solutions. The prepared coating working solutions were stored at 2-8° C. for future use.

[0107] The antibody coating diluent is prepared by adding 1 g of sucrose into 100 mL of 0.05 M pH 8.0 Tris buffer and dissolving and mixing the mixture.

[0108] Step S302, streaking the first coating working solution, the second coating working solution, the third coating working solution, the fourth coating working solution and the fifth coating working solution on the nitrocellulose membrane respectively to form the first detection line, the second detection line, the third detection line, the fourth detection line and the fifth detection line.

[0109] Specifically, the nitrocellulose membrane (NC membrane) was cut into 25 mm×(300±5) mm, the protective film at the NC membrane sticking place on the bottom plate was peeled off, and the NC membrane was evenly pushed from left to right to make it firm.

[0110] Turn on the gold spraying film scribing machine, set the scribing volume to 1 μL / cm, change the scribing length to 300 mm, adjust the scribing needle position, and the line distances between T1 and T2, T2 and T3, T3 and T4, and T4 and T5 are all (3±0.5) mm.

[0111] In some embodiments, the distances between T1 and T2, T3 and T4, and T4 and T5 are all (3±0.5) mm, the distance between T1 line and the lower edge of the nitrocellulose membrane is (11±0.5) mm, and the distance between T3 line and the lower edge of the nitrocellulose membrane is (8±0.5) mm.

[0112] Step S303, preparing a quality control line coating working solution containing animal IgG antibodies.

[0113] Dilute the animal IgG antibody to 1.0-1.5 mg / mL with antibody coating diluent, which is the quality control line coating working solution; the prepared solution is stored at 2-8°C for future use.

[0114] Step S304, streaking the quality control line coating working solution onto the nitrocellulose membrane to form a quality control line.

[0115] Turn on the gold-spraying film scribing machine, set the scribing volume to 1 μL / cm, change the scribing length to 300 mm, and adjust the position of the scribing needle so that the distance between T5 and C lines is (3±0.5) mm.

[0116] In some embodiments, the distance between T2 and line C, and the distance between T5 and line C are both (5±0.5) mm, and the distance between line C and the upper edge of the nitrocellulose membrane is (5±0.5) mm.

[0117] Step S305 , drying the scratched nitrocellulose membrane to obtain a reaction membrane 4 .

[0118] After the film is cut, the NC film is placed in an electric hot air drying oven at a temperature of (50±5)°C and a humidity of ≤30% to dry for (60-72) hours, and is stored at 4-30°C under a relative humidity of ≤30% for later use.

[0119] In some embodiments, the sample pad 2 is prepared by the following preparation steps:

[0120] Prepare sample pad treatment solution;

[0121] The glass fiber is immersed in the sample pad treatment solution and dried to obtain a sample pad 2.

[0122] The components of the sample pad treatment solution include bovine serum, plasma, whole blood albumin, sucrose, 1% (mass ratio) anti-human RBC, 0.1M boric acid borax buffer (pH 8.0) and Tween-20.

[0123] For example, 10 g of bovine serum albumin, 10 g of sucrose and 1% (mass ratio) of anti-human RBC were dissolved in 1000 mL of 0.1 M boric acid borax buffer (pH 8.0), and then 5 mL of Tween-20 was added and stirred until all dissolved to obtain a sample pad treatment solution.

[0124] In some embodiments, the labeling blocking solution includes bovine serum albumin. The preparation process is: dissolve 1 g of bovine serum albumin (BSA) in 10 mL of purified water, and filter through a microporous filter membrane with a pore size of 0.22 μm after fully dissolved.

[0125] The reconstitution solution includes bovine serum albumin, sucrose, casein sodium salt, trehalose and 0.05M pH 8.0 Tris buffer.

[0126] Specifically, 1 g of bovine serum albumin, 10 g of sucrose, 0.5 g of casein sodium salt, and 5 g of trehalose are added to 100 mL of 0.05 M pH 8.0 Tris to dissolve, and then 0.05 mL of Proclin 300 solution is added and mixed.

[0127] Among them, the preparation of 0.05M pH8.0 Tris: dissolve 6.05g Tris in 800mL purified water, adjust the pH to 8.0±0.05 with 1mol / L HCl or 1mol / L NaOH after dissolution, and make up to 1000mL.

[0128] The present application also provides a kit for simultaneously detecting five oral tumor markers, comprising a test card, a chip and a sample diluent, wherein the test card comprises a card shell and a test strip prepared by the preparation method as described above; the test strip is installed in the card shell; and the chip contains standard curve data.

[0129] Assembly of the test card consists of the following steps:

[0130] (1) Under the conditions of temperature (18-26)° C. and humidity ≤30%, the reaction membrane 4 (first reaction membrane 41 or second reaction membrane 42) is laid flat on a work surface, the protective film on the reaction membrane 4 (first reaction membrane 41 or second reaction membrane 42) where the absorption pad 5 (first absorption pad 51 or second absorption pad 52) is adhered is peeled off, the absorption pad 5 (first absorption pad 51 or second absorption pad 52) is adhered thereto, and the other side is partially covered on the reaction membrane 4 (first reaction membrane 41 or second reaction membrane 42) by 2-3 mm, the protective film on the reaction membrane 4 (first reaction membrane 41 or second reaction membrane 42) where the binding pad 3 (first binding pad 31 or second binding pad 32) is adhered to the sample pad 2 (first sample pad 21 or second sample pad 22) is peeled off, and the sample pad 2 (first sample pad 21 or second sample pad 22) is adhered to the sample pad 2 (first sample pad 21 or second sample pad 22) is peeled off. The membrane is adhered to the binding pad 3 (the first binding pad 31 or the second binding pad 32) on its upper end, ensuring that the binding pad 3 (the first binding pad 31 or the second binding pad 32) faces upward, and one end of the binding pad 3 (the first binding pad 31 or the second binding pad 32) covers the reaction membrane 4 (the first reaction membrane 41 or the second reaction membrane 42) by 1 to 2 cm, and the sample pad 2 (the first sample pad 21 or the second sample pad 22) is adhered thereto, ensuring that the lower end of the sample pad 2 (the first sample pad 21 or the second sample pad 22) is aligned with the sample pad pasting position of the base plate 1, and ensuring that the upper end of the sample pad 2 (the first sample pad 21 or the second sample pad 22) covers the binding pad 3 (the first binding pad 31 or the second binding pad 32), and the pasted base plate is placed in an aluminum foil bag for standby use.

[0131] (2) Cut the prepared bottom plate into strips to obtain test strips, and install each test strip into the lower cover of the card shell. After installation, cover the upper cover and place it on the card press. Adjust the height of the card press to 3.45±0.05mm so that the upper and lower covers of the card shell are tightly combined to obtain a test card. See Figure 4 shown.

[0132] In this embodiment, the sample diluent is selected from phosphate buffered saline (PBS buffer), and the preparation steps of the sample diluent are as follows:

[0133] Dissolve 0.5 mL of Proclin 300 in 1000 mL of 0.01 M pH 7.4 PBS buffer and mix well to prepare a sample dilution solution.

[0134] Among them, the preparation of 0.01M pH7.4 PBS buffer is as follows: 8g sodium chloride, 0.2g potassium chloride, 1.14g disodium hydrogen phosphate, and 0.27g potassium dihydrogen phosphate are dissolved in 800mL purified water, and after dissolution, the pH is adjusted to 7.40±0.05 with 1mol / L HCl or 1mol / L NaOH, and the volume is fixed to 1000mL.

[0135] In some embodiments, the sample diluent can be prepared in large quantities. After preparation, it is packaged into EP tubes according to the specification of 0.30 mL / tube, labeled on the tube wall, and stored at 4-30°C. The shelf life is 18 months.

[0136] Based on the above-mentioned kit for simultaneously detecting five oral tumor markers, the present application also provides a method for using the kit for simultaneously detecting five oral tumor markers.

[0137] (1) Applicable sample types

[0138] This application kit is suitable for serum, plasma, and whole blood samples. It is not recommended to use samples with hemolysis, lipemia, or icterus.

[0139] (2) Sample collection:

[0140] Whole blood collection: Collect venous blood from the subject using an anticoagulant tube containing lithium heparin, EDTA-K2 or sodium citrate, and shake the collected blood sample for later use;

[0141] Plasma collection: Collect venous blood from the subject using an anticoagulant tube containing lithium heparin, EDTA-K2 or sodium citrate. After blood collection, plasma should be separated as soon as possible to avoid hemolysis.

[0142] Serum collection: Collect venous blood from the subject using a serum tube or a rapid serum tube containing a coagulant. The serum should be separated as soon as possible after blood collection to avoid hemolysis.

[0143] (3) Sample preservation

[0144] After the sample is collected, it should be used as soon as possible. If it cannot be tested in time, serum / plasma / whole blood can be stored at room temperature (10-30°C) for 3 hours; serum / plasma can be stored at 2°C-8°C for 7 days, whole blood can be stored at 2°C-8°C for 3 days; serum / plasma can be stored at -20±5°C for 90 days. Whole blood samples cannot be frozen.

[0145] It should be noted that refrigerated and frozen samples must be fully dissolved and mixed before testing, and can only be tested after returning to room temperature. Do not freeze and thaw repeatedly.

[0146] When the samples were tested using the kit of the present application, the minimum detection limit of CEA antigen was 2.5 ng / mL; the minimum detection limit of SCC antigen was 1.5 mg / mL; the minimum detection limit of LAP antigen was 500 μg / mL; the minimum detection limit of LSA antigen was 1.7 mg / L; and the minimum detection limit of GST antigen was 2.6 μg / L.

[0147] The kit for simultaneously detecting five oral tumor markers provided by the present application can obtain the same effect as the accuracy and stability of the currently mature and stable radioimmunoassay results, and has higher sensitivity and shorter detection time than radioimmunoassay, simplifies the operation steps of the detection of five oral tumor markers, and saves detection time. The kit only needs to mix the sample to be tested with the sample diluent, add the mixed solution to the test card for 10 minutes, and the results of the five indicators can be obtained without various supporting reagents or expensive instruments. In summary, the kit has the advantages of being fast, sensitive, easy to operate, low cost, and having broad application prospects.

[0148] Obviously, the embodiments described above are only some embodiments of the present application, rather than all embodiments. The preferred embodiments of the present application are given in the accompanying drawings, but they do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive. Although the present application is described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions recorded in the aforementioned specific implementation methods, or to perform equivalent replacement of some of the technical features therein. Any equivalent structure made using the contents of the specification and drawings of this application, directly or indirectly used in other related technical fields, is similarly within the scope of patent protection of this application.

Claims

1. A test strip for simultaneously detecting five oral tumor markers, characterized in that: It includes a sample pad, a binding pad, a reaction membrane and an absorption pad, wherein the sample pad, the binding pad, the reaction membrane and the absorption pad are overlapped on a bottom plate in sequence; The conjugate pad contains colloidal gold-labeled animal anti-human CEA monoclonal antibody, colloidal gold-labeled animal anti-human SCC monoclonal antibody, colloidal gold-labeled animal anti-human LAP monoclonal antibody, colloidal gold-labeled animal anti-human LSA monoclonal antibody and colloidal gold-labeled animal anti-human GST monoclonal antibody; the reaction membrane is provided with a first detection line coated with CEA monoclonal antibody, a second detection line coated with SCC monoclonal antibody, a third detection line coated with LAP monoclonal antibody, a fourth detection line coated with LSA monoclonal antibody, a fifth detection line coated with GST monoclonal antibody and a quality control line coated with animal IgG antibody.

2. The test strip for simultaneously detecting five oral tumor markers according to claim 1, characterized in that: The test strip comprises a first colloidal gold test paper unit and a second colloidal gold test paper unit, the sample pad comprises a first sample pad and a second sample pad, the binding pad comprises a first binding pad and a second binding pad, the reaction membrane comprises a first reaction membrane and a second reaction membrane, and the absorption pad comprises a first absorption pad and a second absorption pad; The first colloidal gold test paper unit is composed of the first sample pad, the first conjugation pad, the first reaction membrane and the first absorption pad which are sequentially overlapped on the bottom plate; the second colloidal gold test paper unit is composed of the second sample pad, the second conjugation pad, the second reaction membrane and the second absorption pad which are sequentially overlapped on the bottom plate; The first conjugate pad contains colloidal gold-labeled animal anti-human CEA monoclonal antibodies and colloidal gold-labeled animal anti-human SCC monoclonal antibodies; the second conjugate pad contains colloidal gold-labeled animal anti-human LAP monoclonal antibodies, colloidal gold-labeled animal anti-human LSA monoclonal antibodies and colloidal gold-labeled animal anti-human GST monoclonal antibodies; the first reaction membrane is provided with a first detection line coated with CEA monoclonal antibodies, a second detection line coated with SCC monoclonal antibodies and a quality control line coated with animal IgG antibodies; the second reaction membrane is provided with a third detection line coated with LAP monoclonal antibodies, a fourth detection line coated with LSA monoclonal antibodies, a fifth detection line coated with GST monoclonal antibodies and a quality control line coated with animal IgG antibodies.

3. The test strip for simultaneously detecting five oral tumor markers according to claim 1, characterized in that: The colloidal gold is selected from nano-gold particles, and the diameter of the nano-gold particles is 90-110nm; the species of the animals include rabbits, sheep, chickens and mice.

4. A method for preparing a test strip for simultaneously detecting five oral tumor markers, characterized in that: The following steps are involved: Prepare sample pads, conjugate pads, reaction membranes and absorption pads; Cover one end of the sample pad with one end of the conjugate pad, cover the other end of the conjugate pad with the end of the reaction membrane away from the quality control line, cover one end of the absorption pad with the other end of the reaction membrane close to the quality control line, and stick them on the bottom plate in sequence to form a test strip; Wherein, the conjugate pad is prepared by the following preparation steps: Prepare colloidal gold solution; Using the colloidal gold solution to label animal anti-human CEA monoclonal antibody, animal anti-human SCC monoclonal antibody, animal anti-human LAP monoclonal antibody, animal anti-human LSA monoclonal antibody and animal anti-human GST monoclonal antibody, respectively, to obtain colloidal gold labeled CEA antibody solution, colloidal gold labeled SCC antibody solution, colloidal gold labeled LAP antibody solution, colloidal gold labeled LSA antibody solution and colloidal gold labeled GST antibody solution; The colloidal gold labeled CEA antibody solution, the colloidal gold labeled SCC antibody solution, the colloidal gold labeled LAP antibody solution, the colloidal gold labeled LSA antibody solution and the colloidal gold labeled GST antibody solution are mixed according to a preset volume ratio to obtain a labeled mixture; spraying the marking mixture onto glass fiber to obtain the conjugate pad; and The reaction membrane is prepared by the following preparation steps: Prepare a first coating working solution containing CEA monoclonal antibody, a second coating working solution containing SCC monoclonal antibody, a third coating working solution containing LAP monoclonal antibody, a fourth coating working solution containing LSA monoclonal antibody, and a fifth coating working solution containing GST monoclonal antibody; The first coating working solution, the second coating working solution, the third coating working solution, the fourth coating working solution and the fifth coating working solution are respectively streaked on the nitrocellulose membrane to form a first detection line, a second detection line, a third detection line, a fourth detection line and a fifth detection line; Prepare the quality control line coating working solution containing animal IgG antibodies; Streaking the quality control line coating working solution onto a nitrocellulose membrane to form a quality control line; The nitrocellulose membrane after the filming is dried to obtain a reaction membrane.

5. The method for preparing a test strip for simultaneously detecting five oral tumor markers according to claim 4, characterized in that: The sample pad is prepared by the following preparation steps: Prepare sample pad treatment solution; The glass fiber is immersed in the sample pad treatment solution and dried to obtain the sample pad.

6. The method for preparing a test strip for simultaneously detecting five oral tumor markers according to claim 4, characterized in that: The colloidal gold solution is prepared by the following preparation steps: Put double distilled water into a beaker and heat to boiling; Add gold chloride aqueous solution and continue heating until boiling; Rapidly add trisodium citrate solution to the boiling gold chloride aqueous solution, continue heating and boiling until the color of the solution changes from black to wine red, stop heating, and obtain a colloidal gold solution; The colloidal gold solution was naturally cooled to room temperature, fixed to volume with purified water, and stored at room temperature for later use.

7. The method for preparing a test strip for simultaneously detecting five oral tumor markers according to claim 4, characterized in that: The steps of using the colloidal gold solution to label animal anti-human CEA monoclonal antibody, animal anti-human SCC monoclonal antibody, animal anti-human LAP monoclonal antibody, animal anti-human LSA monoclonal antibody and animal anti-human GST monoclonal antibody respectively to obtain colloidal gold labeled CEA antibody solution, colloidal gold labeled SCC antibody solution, colloidal gold labeled LAP antibody solution, colloidal gold labeled LSA antibody solution and colloidal gold labeled GST antibody solution include: Take five centrifuge tubes, add colloidal gold solution to each tube, and add potassium carbonate solution to each tube to adjust the pH value; Adding animal anti-human CEA monoclonal antibody, animal anti-human SCC monoclonal antibody, animal anti-human LAP monoclonal antibody, animal anti-human LSA monoclonal antibody and animal anti-human GST monoclonal antibody into different centrifuge tubes, mixing them evenly, and obtaining colloidal gold CEA antibody solution, colloidal gold SCC antibody solution, colloidal gold LAP antibody solution, colloidal gold LSA antibody solution and colloidal gold GST antibody solution respectively; Add the labeled blocking solution to each centrifuge tube for blocking; After the blocking reaction is completed, each centrifuge tube is centrifuged; After centrifugation, the supernatant was discarded, and the precipitate was retained and added to the reconstitution solution to obtain colloidal gold labeled CEA antibody solution, colloidal gold labeled SCC antibody solution, colloidal gold labeled LAP antibody solution, colloidal gold labeled LSA antibody solution and colloidal gold labeled GST antibody solution.

8. The method for preparing a test strip for simultaneously detecting five oral tumor markers according to claim 7, characterized in that: The labeling blocking solution includes bovine serum albumin; The reconstitution solution includes bovine serum albumin, sucrose, casein sodium salt, trehalose and 0.05M pH 8.0 Tris buffer.

9. The method for preparing a test strip for simultaneously detecting five oral tumor markers according to claim 4, characterized in that: The preset volume ratio is 1:1:1:1:1; The concentrations of the first coating working solution, the second coating working solution, the third coating working solution, the fourth coating working solution and the fifth coating working solution are 1.0-1.5 mg / mL respectively; The concentration of the quality control line coating working solution is 1.0-1.5 mg / mL.

10. A kit for simultaneously detecting five oral tumor markers, comprising a detection card, a chip and a sample diluent, characterized in that: The detection card comprises a card housing and a test strip according to any one of claims 1 to 3 or a test strip prepared by the preparation method according to any one of claims 4 to 9; the test strip is installed in the card housing; and the chip contains standard curve data.

Images