WO2022099921A1 - 一种新型免疫层析检测装置 - Google Patents

一种新型免疫层析检测装置 Download PDF

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WO2022099921A1
WO2022099921A1 PCT/CN2021/071116 CN2021071116W WO2022099921A1 WO 2022099921 A1 WO2022099921 A1 WO 2022099921A1 CN 2021071116 W CN2021071116 W CN 2021071116W WO 2022099921 A1 WO2022099921 A1 WO 2022099921A1
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Prior art keywords
sample
pad
pretreatment
detection device
detection
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PCT/CN2021/071116
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English (en)
French (fr)
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张敏
杨蓉
宋学文
王韩梦
郭秀丽
赵师伊
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杭州微策生物技术股份有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • the invention belongs to the technical field of biological detection, and relates to a novel immunochromatographic detection device, in particular to a one-step immunochromatographic detection device including pretreatment for detecting novel coronavirus.
  • the detection of new coronavirus is mainly based on PCR-based viral nucleic acid detection.
  • the detection principle is to use the unique gene sequence of the virus as the detection target, and through PCR amplification, the target DNA sequence we choose increases exponentially. The more each amplified target gene, the stronger the accumulated fluorescent signal. In samples without virus, because there is no target gene amplification, the result of fluorescence signal enhancement cannot be detected.
  • the detection of new coronavirus by nucleic acid has high specificity and sensitivity, but this detection method has high technical requirements, and the specimen needs special treatment, requiring professional equipment such as PCR amplifier and gel electrophoresis. The test takes a long time, and requires professional and technical personnel to operate and judge the test results, and cannot be used for early preliminary screening at the grassroots level such as community hospitals, customs, and CDC.
  • Chinese patent application 202010184358.X and others have disclosed a fluorescence immunochromatographic device for detecting COVID-19 and its use method
  • Chinese patent application 202010184382 and others have disclosed a colloidal gold immunochromatography device and method for detecting COVID-19.
  • the corresponding product can be applied to the early preliminary screening at the grassroots level, but it still has certain operational defects, especially the method for detecting viral antigens requires the first time to dilute or lyse the collected samples for further detection.
  • Two-step detection collect samples from the patient's oropharynx or nasopharynx, process the collected samples in other auxiliary devices other than the detection device after sampling, and then add the processed samples to the detection device for result detection .
  • the present invention provides a new type of immunochromatographic detection device.
  • the immunochromatographic detection device includes a sample pretreatment part, which does not require pretreatment such as dilution or cracking of the sample during use.
  • One-step detection The one-step immunochromatographic detection device including the pretreatment part provided by the present invention can be used for the immunochromatographic detection of the new coronavirus SARS-CoV-2, and realizes the one-step method for diagnosing the negative and positive of the new coronavirus antigen to diagnose the negative and positive of the new coronavirus.
  • Convenient, fast and efficient it is suitable for the initial rapid diagnosis of new coronavirus infection by various medical institutions or individuals.
  • the immunochromatographic detection device can use the oral cavity to directly take saliva samples for sample collection.
  • the oral cavity sampling has less invasiveness and less patient discomfort.
  • Collecting saliva samples is technically less complex and therefore reduces the risk of virus transmission to healthcare workers and health care workers by reducing procedure time and allowing patients to self-collect under supervision. Patients collect themselves under supervision, which also reduces the use of personal protective equipment.
  • the one-step testing method is simpler and more marketable.
  • the present invention provides a novel immunochromatographic detection device, the detection device includes a sample collection site and a test strip, the test strip includes a sample pretreatment pad, and the sample collected in the sample collection site
  • the sample can be delivered to a sample preparation pad that includes dry immobilized pretreatment reagents for viral sample lysis.
  • test strip includes a bottom plate, a sample pretreatment pad, a sample pad, a binding pad, a reaction membrane and a water absorbing pad, wherein the sample pretreatment pad, the sample pad, the binding pad, the reaction membrane and the water absorbing pad are sequentially overlapped together;
  • sample collection site includes a sample collection block and a sampling groove.
  • the sample pretreatment pad, the sample pad, the binding pad, the reaction membrane, and the absorbent pad are sequentially overlapped and positioned on the bottom plate.
  • the sample pretreatment pad, the sample pad, the binding pad, the reaction membrane and the water absorbing pad are overlapped in sequence, the sample pad, the binding pad, the reaction membrane and the water absorbing pad are located on the bottom plate, and the sample pretreatment pad is positioned on the bottom plate. outside the baseplate.
  • the detection device further includes a cartridge in which the test strip is installed, the front end of the cartridge is provided with a sampling slot for accommodating the sample collection block, and a filter component is provided at the connection between the sample collection block and the test strip.
  • the sample collection block is a sponge that can absorb samples, the sample collection block partially exposes the sampling groove, and the exposed part is covered with a sampling cover, which is used to squeeze the sample collection block, so that the sample passes through the filter part and reaches the sample pretreatment pad.
  • the sample sampling block can be sealed therein through the cooperation of the sample sampling cover and the sampling groove.
  • the card case includes a card case cover and a card case bottom, the bottom of the card case is provided with a test paper strip groove, and the front end of the test paper strip groove includes a pretreatment pad groove.
  • the sample pretreatment pad is just in the pretreatment pad slot.
  • the card case cover and the card case bottom are assembled together for accommodating the sample collection block and the test strip; and after the card case cover and the card case bottom are assembled together, the front end forms a sampling groove.
  • the sample pretreatment pad can be placed separately from the test strip, that is, the sample pretreatment pad is placed in the pretreatment pad slot alone, the test strip is placed in the test strip slot, and the test strip sample pad The beginning end overlaps with the end of the pretreatment pad, and this embodiment also falls within the protection scope of the present invention.
  • the filter component is a filter screen
  • the filter screen is located on the cartridge cover and/or the cartridge bottom, and the width of the filter screen is not greater than the width of the sample pretreatment pad.
  • the cartridge cover is provided with a cartridge cover filter screen, the cartridge bottom is provided with a cartridge bottom filter screen, or only one of the two is provided.
  • the sample sampling block is squeezed with the sampling cover, the sample flows through the filter screen to the pretreatment tank area.
  • the width of the filter screen is equivalent to the width of the sample pretreatment pad, so that the sample can be just received by the pretreatment pad after passing through the filter screen without causing leakage and waste.
  • sample collection block is provided with a groove to be fixed in the sampling groove at the front end of the cartridge, and the distance between the sample collection block and the filter screen is 1-3 mm.
  • the distance between the sample collection block and the filter screen is 2-3 mm.
  • the sample is saliva
  • the pretreatment liquid reagent is a lysis reagent for viruses in the saliva sample.
  • the lysis reagents include: 0.01 mol/L phosphate buffer, 3% casein by mass, 1% stearic acid monoethanolamide, 0.01% proclin 300 by mass, wherein phosphate buffer The pH of the liquid is 7.5-8.5.
  • the labeling pad includes colloidal gold or fluorescent labels.
  • preparation method of described pretreatment pad is:
  • sample pad is treated with a sample pad treatment solution
  • sample pad treatment solution includes 0.05M pH 8.0 boric acid buffer, casein, surfactant S17, cholic acid, and EDTA.
  • binding pad is coated with a novel coronavirus NP protein monoclonal antibody labeled with colloidal gold particles (or fluorescent microspheres).
  • reaction membrane is a nitrocellulose membrane
  • a detection line and a quality control line are arranged on the reaction pad in turn along the flow direction of the sample, and the detection line is coated with a novel coronavirus NP protein monoclonal antibody, and the quality control line is coated.
  • the line is coated with goat anti-mouse polyclonal antibody.
  • novel coronavirus NP protein monoclonal antibody is a mouse anti-SARS-CoV-2 nucleocapsid protein monoclonal antibody.
  • the present invention provides a novel coronavirus SARS-CoV-2 antigen immunochromatographic saliva detection kit and detection method, comprising the following steps:
  • the saliva sample enters the pretreatment pad, the analyte in the saliva reacts with the reagent on the pretreatment pad, and then enters the sample pad through capillary action, and then enters the reaction membrane through the binding pad through capillary action, and the reaction complex follows the reaction membrane.
  • Diffusion forward captured by the coating antibody immobilized on the detection line, thereby showing a color signal visible to the naked eye at the coating antibody, or in other embodiments showing an instrument-measurable signal change at the coating antibody
  • the waste liquid of the chromatography reaction was collected by the absorbent paper, and the chromatography reaction was pulled in this direction.
  • the present invention provides the use of stearic acid monoethanolamide for preparing a virus lysis reagent, wherein the lysis reagent comprises: 0.01 mol/L phosphate buffer, 3% phenol by mass fraction Protein, stearic acid monoethanolamide with a mass fraction of 0.5-1.5%, and proclin 300 with a mass fraction of 0.01%, wherein the pH of the phosphate buffer is 7.5-8.5.
  • the lysis reagents include: 0.01 mol/L phosphate buffer, 3% casein by mass, 1% stearic acid monoethanolamide, 0.01% proclin 300 by mass, wherein phosphate buffer The pH of the liquid is 7.5-8.5.
  • virus is a novel coronavirus.
  • the present invention provides a new type of immunochromatographic detection device
  • the immunochromatographic detection device includes a sample pretreatment part, which does not require pretreatment such as dilution or cracking of the sample during use, and can realize one step method detection.
  • the immunochromatographic detection device provided by the present invention is particularly suitable for the immunochromatographic detection of the novel coronavirus SARS-CoV-2, and can directly collect saliva samples from the oral cavity for sample collection, which is convenient for patients to self-sampling and self-detection, and a one-step method for diagnosing the novel coronavirus Positive and negative for coronavirus, easy to use, fast and efficient, reduce the risk of virus transmission to medical staff and health care workers, and reduce the use of personal protective equipment, suitable for various medical institutions or individuals in the initial stage of infection with the new coronavirus Quick diagnosis.
  • Example 1 is a schematic structural diagram of a novel immunochromatographic detection device in Example 1;
  • Fig. 2 is the disassembly diagram of each layer structure of the novel immunochromatographic detection device in Example 1;
  • Fig. 3 is the structure disassembly diagram of the sample collection site of the novel immunochromatographic detection device in Example 1;
  • Fig. 4 is the colloidal gold standard colorimetric card among the embodiment 4;
  • FIG. 5 is a correlation diagram of the signal average value of the detection line T line without the pretreatment pad and with the pretreatment pad in Example 5.
  • Detection means assaying or testing for the presence or absence of a substance or material.
  • substances or materials are, for example, but not limited to, chemical substances, organic compounds, inorganic compounds, metabolites, drugs or drug metabolites, organic tissues or metabolites of organic tissues, nucleic acids, proteins or polymers.
  • detection can also represent the quantity of a test substance or material.
  • Assays also mean immunoassays, chemical assays, enzymatic assays, and the like.
  • the sample used in the detection device includes biological fluid.
  • the initial state of the sample can be liquid, solid or semi-solid, and the solid or semi-solid sample can be transformed into a liquid sample by any suitable method, such as mixing, mashing, macerating, incubating, dissolving, enzymatic hydrolysis, etc., and then Poured into the collection chamber, the test element detects whether the sample contains the analyte.
  • Samples can be taken from humans, animals, plants, nature, etc. Samples taken from the human body, such as blood, serum, urine, cerebrospinal fluid, sweat, lymph, saliva, gastric juice and other liquid samples; feces, hair, keratin, tartar, finger/toenails and other solid or semi-solid samples .
  • Samples taken from plants can be solid samples such as roots, stems, and leaves; liquid or semi-solid samples such as tissue fluid and cell fluid prepared from roots, stems, and leaves.
  • Samples taken from nature can be liquid samples such as rainwater, river water, seawater, and groundwater; solid or semi-solid samples such as soil, rock, ores, and petroleum.
  • the sample described in the present invention is saliva, which is more convenient for self-sampling and detection.
  • a test device generally includes a test element, and the so-called test element refers to a component that can detect the analyte in the sample to be tested.
  • the detection of the analyte by the test element can be based on any technical principle, eg immunological, chemical, electrical, optical, molecular, physical, etc.
  • the test element of the present invention may be one kind or a combination of two or more test elements.
  • the test element has a detection area for displaying the detection result, and after the detection is performed, the detection area displays the detection result.
  • test strips for analysis of analytes (eg, drugs or metabolites indicative of medical conditions) in a sample can be in various formats, such as in the form of immunoassays or chemical assays.
  • the test strips can be analyzed in a non-competitive or competitive mode.
  • the test strip includes a water-absorbing material with a sample application area, a reagent area and a test area. Add the sample to the sample application area and flow to the reagent area by capillary action. In the reagent area, the sample binds to the reagent if the analyte is present. The sample then continues to flow to the detection zone.
  • reagents such as molecules that specifically bind to the analyte, are immobilized in the detection zone. These reagents react with the analyte (if present) in the sample and bind the analyte in this zone, or bind to one of the reagents in the reagent zone.
  • the label used to display the detection signal is present in the reagent zone or separate label zone.
  • a signal is generated if the analyte is present in the sample, and no signal is generated if the analyte is not present.
  • a signal is generated if the analyte is not present in the sample, and no signal is generated if the analyte is present.
  • the test element can be a test paper, and a material that absorbs or does not absorb water can be selected.
  • Test strips can include a variety of materials for liquid sample transfer.
  • One of the test paper materials can be overlaid on another material, such as filter paper overlaid on a nitrocellulose membrane.
  • One area of the test strip can be selected from one or more materials, while the other area can be selected from other different materials or materials.
  • Test strips can be adhered to a support or hard surface to improve the strength of the test strips.
  • the analyte is detected by a signal generating system, such as using one or more enzymes that specifically react with the analyte, using the method of immobilizing the specific binding substance on the detection test paper as described above, and combining one or more enzymes.
  • the composition of the signal generating system is immobilized on the analyte detection area of the test strip.
  • the signal-generating substance may be in the sample application area, the reagent area, or the detection area, or the entire test strip, and the substance may impregnate one or more materials of the test strip.
  • the signal-containing solution is added to the surface of the test paper or one or more materials of the test paper are immersed in the signal-containing solution.
  • the test paper to which the signal containing solution was added was allowed to dry.
  • test strip can be arranged in the following ways: sample addition area, reagent area, detection area, control area, determination of whether the sample is adulterated or not, and liquid sample absorption area.
  • the control area is located after the detection area. All zones can be arranged on a strip of test strips using only one material. It is also possible to use different materials for different zones. Each zone can be in direct contact with the liquid sample, or different zones can be arranged according to the direction of the liquid sample flow, and the end of each zone can be connected and overlapped with the front end of another zone.
  • the material used can be a material with better water absorption such as filter paper, glass fiber or nitrocellulose membrane. Test strips can also take other forms.
  • the detection reagent strip applied to the present invention can be the so-called lateral flow test strip (Lateral flow test strip).
  • Common detection reagent strips include a sample collection area, a labeling area, a detection area and a water absorption area.
  • the sample collection area includes a sample receiving pad, and the labeling area includes a labeling pad.
  • the necessary chemical substances for the analyte such as immunological reagents or enzymatic chemical reagents.
  • the commonly used detection reagent strips are nitrocellulose membrane reagent strips, that is, the detection area includes a nitrocellulose membrane, and specific binding molecules are immobilized on the nitrocellulose membrane to display the detection results; it can also be a cellulose acetate membrane or a nylon membrane, etc.
  • a detection result control area may also be included downstream of the detection area.
  • the control area and the detection area appear in the form of horizontal lines, which are detection lines or control lines.
  • detection reagent strips are traditional reagent strips, of course, other types of reagent strips that utilize capillary action for detection.
  • the general detection reagent strip has dry chemical reagent components, such as immobilized antibodies or other reagents. When encountering liquid, the liquid flows along the reagent strip with capillary action, and with the flow, the dry reagent components are dissolved in the The liquid, thus proceeding to the next zone for processing, reacts with the dry reagents in that zone to perform the necessary detection. Liquid flow occurs primarily through capillary action.
  • FIG. 3 is a structural disassembly diagram of the sample collection site of the novel immunochromatographic detection device.
  • the present invention provides a new type of immunochromatographic detection device.
  • the detection device includes a sample collection site 1 and a test strip 2.
  • the test strip 2 includes a sample pretreatment pad 3.
  • the sample collection site The sample collected in 1 can be transferred to the sample pretreatment pad 3, and the sample pretreatment pad 3 has a dry and fixed sample pretreatment solution.
  • the test strip 1 includes a bottom plate 4 , a sample pretreatment pad 3 , a sample pad 5 , a binding pad 6 , a reaction membrane 7 and a water absorption pad 8 , a sample pretreatment pad 3 , a sample pad 5 , a binding pad 6 , and a reaction membrane 7 . It overlaps with the absorbent pad 8 in sequence and is located on the bottom plate 4 . In some manners, only the sample pad 5 , the bonding pad 6 , the reaction membrane 7 and the water absorption pad 8 can be located on the bottom plate, and the sample pretreatment pad 3 can be located outside the bottom plate 4 .
  • the sample collection site 1 includes a sample collection block 9 and a sampling tank 10 .
  • the detection device further includes a cartridge 11, in which the test strip 2 is installed, the front end of the cartridge 11 is provided with a sampling slot 10 for accommodating the sample collection block 8, and the connection between the sample collection block 9 and the test strip 2 is provided with There is a filter element 12 .
  • the sample collection block 9 is a sponge that can absorb samples.
  • the sample collection block 9 partially exposes the sampling groove 10, and the exposed part is covered with a sampling cover 13, which is used to squeeze the sample collection block 9 so that the sample can pass through the filter element to reach the sample.
  • Pre-treatment pad 3 The sample sampling block 9 can be sealed therein through the cooperation of the sampling cover 13 with the sampling groove 10 .
  • the card case 11 includes a card case cover 14 and a card case bottom 15 , the card case bottom 15 is provided with a test strip slot 16 , the front end of the test strip slot 16 includes a pretreatment pad slot 17 , and the test strip 2 is located in the test strip slot 16 , the sample pretreatment pad 3 is just located in the pretreatment pad groove 17 .
  • the cartridge cover 14 and the cartridge bottom 15 are assembled together for accommodating the sample collection block 9 and the test strip 2 ; and after the cartridge cover 14 and the cartridge bottom 15 are assembled together, the front end forms the sampling slot 10 .
  • the sample pretreatment pad 3 can be placed separately from the test strip 2, that is, the sample pretreatment pad 3 is placed in the pretreatment pad slot 17 alone, the test strip 2 is placed in the test strip slot 16, and the sample pad The starting end of 5 is overlapped with the end of the pretreatment pad 3 .
  • the filter part 12 is a filter screen 18 , the filter screen 18 is located on the cartridge cover 14 and/or the cartridge bottom 15 , and the width of the filter screen 18 is not greater than the width of the sample pretreatment pad 3 .
  • the sample collection block 9 is provided with a groove 19 to be fixed in the sampling groove 10 , and the distance between the sample collection block 9 and the filter screen 18 is 1 ⁇ 3 mm.
  • the distance between the sample collection block 9 and the filter screen 18 is 2 ⁇ 3 mm.
  • the sample is saliva.
  • the sample pretreatment solution is a lysis solution for virus lysis.
  • the virus is a novel coronavirus.
  • the preparation steps of the immunochromatographic detection test device for detecting the novel coronavirus SARS-CoV-2 provided in this embodiment include:
  • Step 1 Prepare the Sample Prep Pad
  • sample pad treatment solution Evenly disperse the sample pad treatment solution on the glass fiber, take it out after drying in a drying oven at 37°C overnight, and then place it in an aluminum foil bag and seal it for later use.
  • the labeled antibody complex solution of 1) is uniformly sprayed on the untreated glass fiber at a certain mass fraction (for example, 2uL/cm) with a gold-spraying instrument.
  • Step 4 Scribing the nitrocellulose membrane
  • Step 5 Assembly Cut and Bag
  • the pretreatment pad in step 5 can also be cut by sticking on the base plate alone, or without the support of the base plate, and the width of the pretreatment pad is greater than or equal to the width of the sample pad.
  • Example 3 Preparation of another immunochromatographic detection device (colloidal gold method) for detecting novel coronavirus
  • Example 2 Same as Example 2, its difference is to use stearic acid monoethanolamide to replace Triton X-100 in the preparation of the pretreatment pad, and the remaining components are the same as other contents in Example 2, and are based on the quality of stearic acid monoethanolamide.
  • the scores differ and are divided into three formulations for the preparation of pretreatment pads.
  • Formulation 1 0.01mol/L phosphate buffer, 3% casein, 0.5% stearic acid monoethanolamide, 0.01% proclin 300.
  • Formulation 2 0.01mol/L phosphate buffer, 3% casein, 1% stearic acid monoethanolamide, 0.01% proclin 300.
  • Formulation 3 0.01mol/L phosphate buffer, 3% casein by mass, 1.5% mass fraction of stearic acid monoethanolamide, and 0.01% mass fraction of proclin 300.
  • the preparation steps of the immunochromatographic detection test device (fluorescence method) for detecting the novel coronavirus SARS-CoV-2 provided by the present embodiment include:
  • Step 1 Preparing the Pretreatment Pad
  • sample pad treatment solution Evenly disperse the sample pad treatment solution on the glass fiber, take it out after drying in a drying oven at 37°C overnight, and then place it in an aluminum foil bag and seal it for later use.
  • the antibody marker complex solution in 1) was sprayed on the glass fiber in 4) with a certain mass fraction (for example, 1uL/cm) with a gold-spraying instrument, placed in a drying oven at 37°C for overnight drying, and then taken out. Seal in aluminum foil bag and set aside.
  • Step 4 Scribing the nitrocellulose membrane (reaction membrane)
  • Step 5 Assembly Cut and Bag
  • the pretreatment pad in step 5 can also be cut by sticking on the base plate alone, or without the support of the base plate, and the width of the pretreatment pad is greater than or equal to the width of the sample pad.
  • Example 4 The same as Example 4, the difference is that Triton X-100 in the preparation of the pretreatment pad is replaced by stearic acid monoethanolamide, and the following three formulas are configured respectively according to the difference of its mass fraction, and other components are the same as in Example 4:
  • Formulation A 0.01mol/L phosphate buffer, 3% casein, 0.5% stearic acid monoethanolamide, 0.01% proclin 300.
  • Formulation B 0.01mol/L phosphate buffer, 3% casein, 1% stearic acid monoethanolamide, 0.01% proclin 300.
  • Formulation C 0.01mol/L phosphate buffer, 3% casein, 1.5% stearic acid monoethanolamide, 0.01% proclin 300.
  • Example 6 Validation of the immunochromatographic detection device for the detection of novel coronavirus prepared by the colloidal gold method provided in Example 2
  • the dilution times are greater than 6400 times. After that, a negative result will appear when interpreting the T-line signal of the detection line.
  • a standard colorimetric card see Figure 4 for comparison.
  • test result is positive; if the test line T ⁇ G3, the test result is negative, and the quality control line is always C ⁇ G8; color, even if the detection line T develops color, the test result will be invalid.
  • the first group is to directly use the saliva detection kit without pretreatment pad (without sample lysis) (see Table 1 for the specific results)
  • the second group is to first perform sample lysis treatment and then use without pretreatment.
  • Treatment pad saliva detection kit see Table 2 for specific results
  • the third group is to use the saliva detection kit with pretreatment pad provided in Example 2 for detection (see Table 3 for specific results), and each treatment is repeated 20 times
  • Use a standard color chart to interpret the test results (G represents the depth of the color to interpret the test results).
  • Groups 1, 2 and 3 have the same test strip construction and reagents, except for the following differences: the first group has no pretreatment; the second group has pretreatment, but the test strips are still traditional tests without pretreatment pads The third group is the test strips including the pretreatment pads.
  • the detection device is rewarmed at 15-30°C.
  • the diluted virus culture was added to the saliva samples according to the gradient in 2), and a total of 8 samples were obtained. 1/1600, 1/800, 1/400, 1/200, 1/100, in addition to 1 sample, no virus culture was added, and the culture concentration was 0.
  • test results are shown in Table 1, Table 2, and Table 3.
  • the first group shows that the results are obviously weak, and it is difficult to detect the real results (without cracking); the color development of the second and third groups
  • the results are consistent, and there is no difference in chromaticity. Therefore, it is feasible to use the detection device of the present invention for virus detection.
  • a pretreatment pad is set in front of the sample pad on the test strip, and the lysis reagent is included on it, which can truly realize that no need for sample preparation.
  • Pre-treatment such as lysis is performed, and the virus is directly detected by one-step method.
  • Such a detection device does not require additional pretreatment steps, and is directly added dropwise to the test strip, which reduces steps and reduces possible secondary pollution to operators caused by the pretreatment steps.
  • the dilution times are greater than 6400 times. After that, a negative result will appear when interpreting the T-line signal of the detection line.
  • a standard colorimetric card see Figure 4 for comparison.
  • test result is positive; if the test line T ⁇ G3, the test result is negative, and the quality control line is always C ⁇ G8; color, even if the detection line T develops color, the test result will be invalid.
  • the detection devices for preparing the pretreatment pads using formula 1, formula 2, and formula 3 are divided into three groups, wherein formula 1 corresponds to the fourth group (0.5%), and formula 2 corresponds to the fifth group (1%), Formulation 3 corresponds to the sixth group (1.5%).
  • formula 1 corresponds to the fourth group (0.5%)
  • formula 2 corresponds to the fifth group (1%)
  • Formulation 3 corresponds to the sixth group (1.5%).
  • the same saliva samples as in Example 6 were collected for testing, and each group was repeated 20 times, and a standard colorimetric card was used to interpret the testing results.
  • Table 4 sample directly adds the result that the formula 1 detection device of embodiment 3 detects
  • Example 8 Verification of the immunochromatographic detection device for the detection of novel coronavirus prepared by the fluorescence method provided in Example 4
  • T-line signal value was similar to the background value, and it was judged as negative.
  • the first group is to first perform sample lysis treatment and then use the saliva detection kit without pretreatment pad
  • the second group is to use the saliva detection kit with pretreatment pad provided in Example 4 for detection, repeating 3 times, using a fluorescence immunoassay analyzer to detect, the specific experimental process is as follows:
  • the detection device is rewarmed at 15-30°C.
  • the concentration of the virus culture in the samples is 1/500. , 1/1000, 1/2000, 1/4000, 1/5000, 1/7500, 1/10000, 1/12000 of saliva samples, in addition to 1 sample without virus culture, its culture Concentration is 0.
  • the test results are shown in Table 7, Table 8, and Table 9.
  • the C line signal of the quality control line of the two groups of test results is basically stable.
  • the T line signal of the test line is basically similar, and the T line signal of the two groups of data is taken.
  • the pretreatment pad set before the sample pad of the test strip includes a lysis reagent, which can obtain the same effect as independent lysis.
  • Table 8 The present invention has the detection results of the pretreatment pad detection device
  • Example 9 Verification of another immunochromatographic detection device for detecting novel coronavirus prepared by fluorescence method provided in Example 5
  • T-line signal value was similar to the background value, and it was judged as negative.
  • the detection devices for preparing pretreatment pads using formula A, formula B, and formula C are divided into three groups, formula A corresponds to group I, formula B corresponds to group II, and formula C corresponds to group III.
  • the same saliva samples as in Example 8 were collected for detection, repeated 3 times, and detected using a fluorescence immunoassay analyzer.
  • Test result is shown in table 10, table 11, table 12, when Triton X-100 is replaced with stearic acid monoethanolamide, also can detect signal when dilute concentration is 1/12000, compared with embodiment The detection sensitivity in 8 is higher, and when the current treatment pad is prepared by formula B, that is, when the content of stearic acid monoethanolamide is 1%, the positive rate of the detection result is the highest, and the virus is released more completely.
  • Table 10 Table 11
  • Table 12 when Triton X-100 is replaced with stearic acid monoethanolamide, also can detect signal when dilute concentration is 1/12000, compared with embodiment The detection sensitivity in 8 is higher, and when the current treatment pad is prepared by formula B, that is, when the content of stearic acid monoethanolamide is 1%, the positive rate of the detection result is the highest, and the virus is released more completely.

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Abstract

一种免疫层析检测装置，免疫层析检测装置包含样品前处理部分，使用时无需对样品进行稀释或裂解等前处理，可实现一步法检测。免疫层析检测装置适用于新型冠状病毒SARS-CoV-2的快速检测，可采用口腔直接采取唾液样本的方式进行样本采集，方便患者自我采样、自行检测，一步法确诊新型冠状病毒的阴阳性，使用方便，简单高效，降低病毒向医护人员和卫生保健工作者传播的风险，也减少了个人防护装备的利用，适合各类医疗机构或个人对新型冠状病毒感染情况的初期快速诊断。

Description

一种新型免疫层析检测装置 技术领域
本发明属于生物检测技术领域，涉及一种新型免疫层析检测装置，尤其涉及一种用于检测新型冠状病毒的包含前处理的一步法免疫层析检测装置。
背景技术
目前针对新型冠状病毒的检测主要是以PCR为基础的病毒核酸检测，检测原理就是以病毒独特的基因序列为检测靶标，通过PCR扩增，使我们选择的这段靶标DNA序列指数级级增加，每一个扩增的靶基因越多累积的荧光信号越强。而没有病毒的样本中，由于没有靶基因扩增，因此就检测不到荧光信号增强的结果。通过核酸检测新型冠状病毒有较高的特异性和灵敏度，但是该检测方法对技术要求高，标本需要特殊处理，要求具备PCR扩增仪及凝胶电泳等专业的仪器设备，对新型冠状病毒的检测用时长，需要专业技术人员操作和判断检测结果，无法应用于社区医院、海关、疾控中心等基层的早期初步筛查。
目前针对以上问题已经有中国专利申请202010184358.X等公开了检测COVID-19的荧光免疫层析装置及其使用方法，有中国专利申请202010184382等公开了检测COVID-19的胶体金免疫层析装置及使用方法，对应的产品可以应用于基层的早期初步筛查，但是其仍然存在一定的操作缺陷，特别是检测病毒抗原的方法需要首次对采集到的样本进行稀释或裂解才能进一步进行检测，即采用两步法检测，从患者口咽或鼻咽处采集样本，采样后先在检测装置以外的其他辅助装置中将采集到的样本进行处理，再将处理后的样本添加到检测装置中进行结果检测。这种采样可能增加病毒传播给缺乏足够个人防护装备的医护人员和卫生保健工作者的风险，操作步骤不是最简化的，容易造成操作误差，从而对检测结果产生一定的影响。因此急需一种更适于患者自我采集、自行检测的，更简易方便的新型冠状病毒的免疫层析检测装置。
发明内容
为弥补已有技术的缺陷，本发明提供了一种新型的免疫层析检测装置，所述免疫层析检测装置包含样品前处理部分，使用时无需对样品进行稀释或裂解等前处理，可实现一步法检测。本发明提供的包含前处理部分的一步法免疫层析检测装置可用于新型冠状病毒SARS-CoV-2免疫层析检测，实现一步法诊断新冠抗原的阴阳性来确诊新型冠状病毒的阴 阳性，使用方便，快速高效，适合各类医疗机构或个人对新型冠状病毒感染情况的初期快速诊断。
本发明提供的免疫层析检测装置可采用口腔直接采取唾液样本的方式进行样本采集，与其他上呼吸道部位的取样相比，口腔取样具有较小的侵入性和较少的病人不适。采集唾液样本技术上不太复杂，因此可以通过缩短操作时间和允许患者在监督下进行自我采集，从而降低病毒向医护人员和卫生保健工作者传播的风险。患者在监督下进行自我收集，也减少了个人防护装备的利用，相较于现有的方式，一步完成检测的方式更简单也更有市场需求。
本发明提供的技术方案为：
一方面，本发明提供了一种新型的免疫层析检测装置，所述检测装置包括样本采集部位和试纸条，所述试纸条上包含样品前处理垫，所述样本采集部位中采集的样品可传递至样品前处理垫，样品前处理垫上包括干燥固定的用于病毒样本裂解的前处理试剂。
进一步地，所述试纸条包括底板、样品前处理垫、样本垫、结合垫、反应膜和吸水垫，其中，所述的样品前处理垫、样本垫、结合垫、反应膜和吸水垫依次搭接在一起；所述样本采集部位包括样本采集块和采样槽。
在有些实施方式中，样品前处理垫、样本垫、结合垫、反应膜和吸水垫依次搭接在一起并位于底板之上。
还有些实施方式中，样品前处理垫、样本垫、结合垫、反应膜和吸水垫依次搭接在一起，样本垫、结合垫、反应膜和吸水垫位于底板之上，样品前处理垫位与底板之外。
进一步地，所述检测装置还包括卡壳，试纸条装于卡壳内，卡壳前端设有容纳样本采集块的采样槽，样本采集块与试纸条的连接处设有过滤部件。
进一步地，所述样本采集块为可吸附样品的海绵，样本采集块部分露出采样槽，露出部分套有采样盖，用于挤压样本采集块，使样品通过过滤部件到达样品前处理垫。通过样本采样盖与采样槽的配合可将样本采样块密封在其中。
进一步地，所述卡壳包括卡壳盖和卡壳底，卡壳底设有试纸条槽，试纸条槽前端包括前处理垫槽。试纸条位于试纸条槽时，样品前处理垫正好位于前处理垫槽。
卡壳盖和卡壳底组装在一起，用于容纳样本采集块和试纸条；并且卡壳盖和卡壳底组装在一起后，前端形成采样槽。
在有些实施方式中，样品前处理垫可以与试纸条分开放置，即将样品前处理垫单独放置在前处理垫槽中，试纸条放置在试纸条槽中，试纸条样本垫的起始端与前处理垫的末端相搭，这种实施方式也属于本发明的保护范围内。
进一步地，所述过滤部件为过滤网，所述过滤网位于卡壳盖和/或卡壳底上，过滤网的宽度不大于样品前处理垫的宽度。
卡壳盖上设有卡壳盖过滤网，卡壳底上设有卡壳底过滤网，或者两者上只设其一，当使用采样盖挤压样本采样块时，样本通过滤网流到前处理槽区域，滤网的宽度与样品前处理垫的宽度相当，使样本通过滤网后能刚好被前处理垫接收而不造成泄露和浪费。
进一步地，样本采集块上设有凹槽，使其固定于卡壳前端的采样槽内，样本采集块与过滤网之间距离1～3mm。
进一步地，样本采集块与过滤网之间距离2～3mm。
进一步地，所述样品为唾液，所述前处理液试剂为用于唾液样品中的病毒的裂解试剂。
进一步地，所述裂解试剂包括：0.01mol/L的磷酸盐缓冲液、质量分数3％的酪蛋白、质量分数0.5-1.5％的硬脂酸单乙醇酰胺、质量分数0.01％的proclin 300，其中磷酸盐缓冲液的PH＝7.5-8.5。
进一步，所述裂解试剂包括：0.01mol/L的磷酸盐缓冲液、质量分数3％的酪蛋白、质量分数1％的硬脂酸单乙醇酰胺、质量分数0.01％的proclin 300，其中磷酸盐缓冲液的PH＝7.5-8.5。
进一步地，所述的标记垫包括胶体金或者荧光标记。
进一步地，所述前处理垫的制备方式为：
1)配置磷酸盐缓冲液，并将PH调节至7.5-8.5；
2)取一定质量分数的上述磷酸盐缓冲液(例如0.01-0.05mol/L)、一定质量分数的酪蛋白(例如3％-5.5％)、一定质量分数的Triton X-100(例如0.01％-1％)、一定质量分数的proclin 300(例如0.01％-0.1％)配置成样品前处理液；3)将处理液均匀分散在玻璃纤维上，37℃干燥箱中干燥过夜，取出置于铝箔袋内封口备用。
进一步地，所述样本垫通过样本垫处理液处理，所述样本垫处理液包括，0.05M pH 8.0硼酸缓冲液、酪蛋白、表面活性剂S17、胆酸、EDTA。
进一步地，所述结合垫上包被有胶体金颗粒(或荧光微球)标记的新型冠状病毒NP蛋白单克隆抗体。
进一步地，所述反应膜为硝酸纤维素膜，反应垫上沿样品流动方向依次设有检测线和 质控线，所述检测线上包被有新型冠状病毒NP蛋白单克隆抗体，所述质控线上包被有山羊抗鼠多克隆抗体。
进一步地，所述新型冠状病毒NP蛋白单克隆抗体为鼠抗SARS-CoV-2核衣壳蛋白单克隆抗体。
另一方面，本发明提供一种新型冠状病毒SARS-CoV-2抗原免疫层析唾液法检测试剂盒及检测方法，包括以下步骤：
1)将检测试剂盒恢复至室温，从密封的铝箔袋中拿出一个检测装置，轻轻拔开采样盖，露出样本采样块前端。
2)将样本采集块前端放入口腔，含3～5min。
3)盖上采集盖，在水平方向上按压盖子1～2次。
4)唾液样本进入前处理垫，唾液中的待测物与前处理垫上的试剂发生反应，随后通过毛细作用进入样本垫，再通过毛细作用经过结合垫进入反应膜，反应复合物沿着反应膜向前扩散，被固定在检测线上的包被抗体捕获，从而在包被抗体处显示出肉眼可见的颜色信号，或者在其他实施例中在包被抗体处显示出仪器可测的信号变化，吸水纸收集层析反应的废液，拉动层析反应往该方向进行。
5)15～20min通过检视窗判读测试结果。若质控线C和检测线T均显紫红色，则结果为阳性；若只有质控线C显紫红色，检测线T不显色，则结果为阴性；若质控线C和检测T均不显紫红色，则结果无效；若质控线C未显示紫红色，即使检测线T显示紫红色，测试结果也无效。或者在其他实施例中仪器检测检测线T与质控线C的信号，两者换算达到一定数值时即判定为阳性。
再一方面，本发明提供了硬脂酸单乙醇酰胺在用于制备病毒裂解试剂的用途，其特征在于，所述裂解试剂包括：0.01mol/L的磷酸盐缓冲液、质量分数3％的酪蛋白、质量分数0.5-1.5％的硬脂酸单乙醇酰胺、质量分数0.01％的proclin 300，其中磷酸盐缓冲液的PH＝7.5-8.5。
进一步，所述裂解试剂包括：0.01mol/L的磷酸盐缓冲液、质量分数3％的酪蛋白、质量分数1％的硬脂酸单乙醇酰胺、质量分数0.01％的proclin 300，其中磷酸盐缓冲液的PH＝7.5-8.5。
进一步地，所述病毒为新型冠状病毒。
本发明的有益效果为：本发明提供了一种新型的免疫层析检测装置，所述免疫层析检测装置包含样品前处理部分，使用时无需对样品进行稀释或裂解等前处理，可实现一步法 检测。本发明提供的免疫层析检测装置特别适用于新型冠状病毒SARS-CoV-2免疫层析检测，可采用口腔直接采取唾液样本的方式进行样本采集，方便患者自我采样、自行检测，一步法确诊新型冠状病毒的阴阳性，使用方便，快速高效，降低病毒向医护人员和卫生保健工作者传播的风险，也减少了个人防护装备的利用，适合各类医疗机构或个人对新型冠状病毒感染情况的初期快速诊断。
附图说明
图1为实施例1中的新型免疫层析检测装置的结构示意图；
图2为实施例1中的新型免疫层析检测装置的各层结构拆分图；
图3为实施例1中的新型免疫层析检测装置的样品采集部位结构拆分图；
图4为实施例4中的胶体金标准比色卡；
图5为实施例5中的无前处理垫和有前处理垫检测线T线的信号平均值相关性图。
详细说明
下面对本发明涉及的结构或技术术语做进一步的说明，如果没有特别指明，按照本领域的通用的一般术语进行理解和解释。这些说明仅仅是采用举例的方式进行说明本发明的方式是如何实现的，并不能对本发明构成任何的限制，本发明的范围由权利要求进行限定和表达。
检测
检测表示化验或测试一种物质或材料是否存在。所述的物质或材料例如、但并不限于化学物质、有机化合物、无机化合物、新陈代谢产物、药物或者药物代谢物、有机组织或有机组织的代谢物、核酸、蛋白质或聚合物。另外，检测还可以表示测试物质或材料的数量。化验还表示免疫检测、化学检测、酶检测等。
样品
本发明中，检测装置所使用的样品包括生物液体。样本的初始状态可以是液态、固态或半固态的，固态或半固态的样本可以通过任何适当的方法转化成液态样本，例如混合、捣碎、浸软、孵育、溶解、酶解等等，然后倒入收集腔中，通过测试元件检测样本是否含有被分析物。样本可以取自人体、动物、植物、自然界等。取自人体的样本，例如可以是血液、血清、尿液、脑脊髓液、汗液、淋巴液、唾液、胃液等液态样本；粪便、毛发、角 质、牙垢、指/趾甲等固态或半固态的样本。取自植物的样本，例如可以是根、茎、叶等固态样本；由根、茎、叶制备的组织液、细胞液等液态或半固态样本。取自自然界的样本，例如可以是雨水、河水、海水、地下水等液态样本；土壤、岩石、矿石、石油等固态或半固态样本。
在一些方式中，本发明所述的样品为唾液，更方便自我采样检测。
测试装置
测试装置中一般包括测试元件，所谓的测试元件指的是能够对待测样本中的被分析物质进行检测的部件。测试元件对被分析物质的检测可以基于任何技术原理，例如免疫学、化学、电学、光学，分子学、物理学等。本发明的测试元件可以是一种，也可以是两种以上测试元件的组合。测试元件具有用于显示检测结果的检测区，进行检测之后、检测区显示检测结果。
各种测试元件可以被组合在一起运用到本发明中。一种形式是检测试纸。用于分析样本中的被分析物(如毒品或表明身体状况的代谢物)的检测试纸可以是各种形式，如免疫测定或化学分析的形式。检测试纸可以采用非竞争法或竞争法的分析模式。检测试纸包含一具有样本加样区的吸水材料，试剂区和测试区。加样本至样本加样区，通过毛细管作用流到试剂区。在试剂区，如果存在被分析物，样本与试剂结合。然后样本继续流动到检测区。另一些试剂，如与被分析物特异性结合的分子被固定在检测区。这些试剂与样本中的被分析物(如果存在)反应并将被分析物结合在该区，或者与试剂区的某一个试剂结合。用于显示检测信号的标记物存在与试剂区或分离的标记区。
典型的非竞争法分析模式是如果样本中含有被分析物，信号就会产生，如果不包含被分析物，就不产生信号。在竞争法中，如果被分析物不存在于样本中，信号产生，如果存在被分析物，则不产生信号。
测试元件可以是检测试纸，可以选用吸水或不吸水的材料。检测试纸可包括多种材料用于液体样本传递。其中一种检测试纸的材料可覆盖在另一种材料上，如滤纸覆盖在硝酸纤维素膜上。检测试纸的一个区可以选用一种或多种材料，而另一区选用其他不同的一种或多种材料。检测试纸可以被黏附在某种支持物或者硬质表面用于提高拿捏检测试纸的强度。
被分析物通过信号发生系统而被检测到，如利用与本分析物发生特异性反应的一种或多种酶，利用如前述将特异结合物质固定在检测试纸上的方法，将一种或多种信号发生系 统的组合物固定在检测试纸的被分析物检测区。产生信号的物质可在加样区，试剂区，或检测区，或整个检测试纸上，该物质可以充满检测试纸的一种或多种材料上。将含有信号物的溶液加到试纸的表面或将试纸的一种或多种材料浸没在含信号物的溶液中。使加入含信号物溶液的试纸干燥。
检测试纸的各个区可以按以下方式排列：加样区，试剂区，检测区，控制区，确定样本是否掺假区，液体样本吸收区。控制区位于检测区之后。所有的区可以被安排在只用一种材料的一条试纸上。也可是不同区采用不同的材料。各个区可以直接和液体样本接触，或不同的区依据液体样本流动的方向排列，将各区的末端与另一区的前端相连并交叠。所用的材料可以是吸水性较好的材料如滤纸，玻纤或者硝酸纤维素膜等。检测试纸也可以采用其他形式。
运用到本发明的检测试剂条可以是通常所说的横向侧流试剂条(Lateral flow test strip)，这些检测试剂条的具体结构和检测原理在现有技术中是本领域一般技术人员公知的技术。普通的检测试剂条，包括样本收集区域，标记区域，检测区域和吸水区域，样本收集区域包括样本接受垫，标记区域包括标记垫，吸水区域可以包括吸水垫，其中检测区域上包括能检测是否含有被分析物质的必要化学物质,例如免疫试剂或者酶化学试剂。一般常用的检测试剂条为硝酸纤维素膜试剂条，即检测区域包括硝酸纤维素膜，在硝酸纤维素膜上固定特异结合分子来显示检测的结果；还可以是醋酸纤维素膜或尼龙膜等等，当然，在检测区域的下游还可以包括检测结果控制区域，通常，控制区域和检测区域上以横线的形式出现，为检测线或者控制线。这样的检测试剂条是传统的试剂条，当然，也可是其他利用毛细作用进行检测的其它类型的试剂条。另外，一般检测试剂条上带有干化学试剂成分，例如固定的抗体或者其他试剂，当遇到液体后，液体随着毛细作用沿着试剂条流动，随着流动，让干的试剂成分溶解于液体，从而到下一个区域处理在该区的干试剂发生反应，从而进行必要的检测。液体流动主要通过毛细作用进行的。这些测试元件见如下文件的描述和记载：李福刚的《硝酸纤维素膜的再生处理及其吸附蛋白能力的研究》；马红艳，李强等的《胶体金诊断试剂盒中层析膜材料性能的分析》；王勇，王路海等的《一种新型胶体金免疫层析试纸条》。在这里都可以被运用到本发明的检测装置中，或者被设置在检测腔中与液体样本接触，或者用来检测进入检测腔中的液体样本中被分析物质是否存在或者存在的数量。
具体实施方式
下面结合实施例对本发明作进一步详细描述，需要指出的是，以下所述实施例旨在便于对本发明的理解，而对其不起任何限定作用。本实施例中未特别指出的试剂均为已知产品，通过购买市售产品获得。
实施例1本发明提供的新型免疫层析检测装置
本实施例提供的新型免疫层析检测装置如图1-3所示，其中图1为新型免疫层析检测装置的结构示意图，图2为新型免疫层析检测装置的各层结构拆分图，图3为新型免疫层析检测装置的样品采集部位结构拆分图。
如图1，本发明提供了一种新型的免疫层析检测装置，所述检测装置包括样本采集部位1和试纸条2，所述试纸条2上包含样品前处理垫3，样本采集部位1中采集的样品可传递至样品前处理垫3，样品前处理垫3上有干燥固定的样品前处理液。
优选地，试纸条1包括底板4、样品前处理垫3、样本垫5、结合垫6、反应膜7和吸水垫8，样品前处理垫3、样本垫5、结合垫6、反应膜7和吸水垫8依次搭接在一起并位于底板4之上。在有些方式中，也可以仅使样本垫5、结合垫6、反应膜7和吸水垫8位于底板之上，样品前处理垫3位与底板4之外。所述样本采集部位1包括样本采集块9和采样槽10。
优选地，所述检测装置还包括卡壳11，试纸条2装于卡壳11内，卡壳11前端设有容纳样本采集块8的采样槽10，样本采集块9与试纸条2的连接处设有过滤部件12。
优选地，所述样本采集块9为可吸附样品的海绵，样本采集块9部分露出采样槽10，露出部分套有采样盖13，用于挤压样本采集块9，使样品通过过滤部件到达样品前处理垫3。通过采样盖13与采样槽10的配合可将样本采样块9密封在其中。
优选地，所述卡壳11包括卡壳盖14和卡壳底15，卡壳底15设有试纸条槽16，试纸条槽16前端包括前处理垫槽17，试纸条2位于试纸条槽16时，样品前处理垫3正好位于前处理垫槽17。卡壳盖14和卡壳底15组装在一起，用于容纳样本采集块9和试纸条2；并且卡壳盖14和卡壳底15组装在一起后，前端形成采样槽10。在有些方式中，样品前处理垫3可以与试纸条2分开放置，即将样品前处理垫3单独放置在前处理垫槽17中，试纸条2放置在试纸条槽16中，样本垫5的起始端与前处理垫3的末端相搭。
优选地，所述过滤部件12为过滤网18，所述过滤网18位于卡壳盖14和/或卡壳底15上，过滤网18的宽度不大于样品前处理垫3的宽度。
优选地，样本采集块9上设有凹槽19，使其固定于采样槽10内，样本采集块9与过滤网18之间距离1～3mm。
优选地，样本采集块9与过滤网18之间距离2～3mm。
优选地，所述样品为唾液。
优选地，所述样品前处理液为用于裂解病毒的裂解液。
优选地，所述病毒为新型冠状病毒。
实施例2检测新型冠状病毒的免疫层析检测装置(胶体金法)的制备
本实施例提供的检测新型冠状病毒SARS-CoV-2的免疫层析检测试装置(胶体金法)的制备步骤包括：
步骤一：制备样品前处理垫
1)配置磷酸盐缓冲液，并将PH调节至7.5。
2)取一定质量分数的上述磷酸盐缓冲液：0.01mol/L、一定质量分数的酪蛋白：3％、一定质量分数的Triton X-100：1％、一定质量分数的proclin 300：0.01％配置成前处理液。
3)将前处理液均匀分散在玻璃纤维上，37℃干燥箱中干燥过夜，取出置于铝箔袋内封口备用。
步骤二：样本垫处理
1)配置硼酸缓冲液，并将PH调节至7.5。
2)取一定质量分数的硼酸缓冲液：0.01mol/L)、一定质量分数的酪蛋白：0.1％，一定质量分数的表面活性剂S17：1％；一定质量分数的胆酸：0.1％，一定质量分数的EDTA：0.6％配置成样本垫处理液。
3)将样本垫处理液均匀分散在玻璃纤维上，37℃干燥箱干燥过夜后取出，再置于铝箔袋内封口备用。
步骤三：结合垫处理
1)将胶体金溶液的PH调节至9.0，与新型冠状病毒NP蛋白单克隆抗体混匀反应，得到标记抗体，用复溶液复溶。
2)将1)的标记抗体复溶液用划膜喷金仪以一定质量分数(例如2uL/cm)均匀喷涂在未处理的玻璃纤维上。
3)将喷涂后的玻璃纤维置于37℃干燥箱过夜干燥，取出后置于铝箔袋封口待用。
步骤四：硝酸纤维素膜划膜
1)配置磷酸盐缓冲液，并将PH调节至8。
2)取一定质量分数的磷酸盐缓冲液(例如0.1mol/L)将新型冠状病毒NP蛋白单克隆抗体稀释到1.0mg/ml,作为T线溶液。
3)取一定质量分数的磷酸盐缓冲液(例如0.1mol/L)将山羊抗鼠多克隆抗体稀释到0.5mg/ml，作为C线溶液。
4)用划膜喷金仪以一定质量分数(如0.5uL/cm)进行划膜。
5)将划好的膜置于37℃干燥箱干燥过夜后取出，置于铝箔袋封口待用。
步骤五：组装切割与装袋
1)将步骤一得到的前处理垫，步骤二处理好的样本垫，步骤三得到的结合垫，步骤四处理好的硝酸纤维素膜，吸水纸裁切为适当宽度，依次黏在底板上，每层之间搭牢1-2mm，前处理垫的宽度大于或等于样本垫的宽度。
2)将粘好的大卡用斩切机斩切成3-6mm宽测试卡，装进卡壳中。
3)将样本采集块固定在卡壳前端。
4)盖上样本采集盖。
5)装入铝箔袋中密封保存。
优选地，步骤五中的前处理垫也可以单独黏在底板上裁切，或者无需底板支撑，前处理垫的宽度大于或等于样本垫的宽度。
实施例3：另一种检测新型冠状病毒的免疫层析检测装置(胶体金法)的制备
与实施例2相同，其区别是使用硬脂酸单乙醇酰胺替代前处理垫制备中的Triton X-100，其余成分与实施例2中的其它含量相同，并根据硬脂酸单乙醇酰胺的质量分数不同，分成三个配方用于制备前处理垫。
配方1：0.01mol/L的磷酸盐缓冲液、质量分数3％的酪蛋白、质量分数0.5％的硬脂酸单乙醇酰胺、质量分数0.01％的proclin 300。
配方2：0.01mol/L的磷酸盐缓冲液、质量分数3％的酪蛋白、质量分数1％的硬脂酸单乙醇酰胺、质量分数0.01％的proclin 300。
配方3：0.01mol/L的磷酸盐缓冲液、质量分数3％的酪蛋白、质量分数1.5％的硬脂酸单乙醇酰胺、质量分数0.01％的proclin 300。
实施例4：检测新型冠状病毒的免疫层析检测装置(荧光法)的制备
本实施例提供的检测新型冠状病毒SARS-CoV-2的免疫层析检测试装置(荧光法)的 制备步骤包括：
步骤一：制备前处理垫
1)配置磷酸盐缓冲液，并将PH调节至8.5。
2)取一定质量分数的上述磷酸盐缓冲液：0.01mol/L、一定质量分数的酪蛋白：3％、一定质量分数的Triton X-100：1％、一定质量分数的proclin 300：0.01％配置成前处理液。
3)将前处理液均匀分散在玻璃纤维上，37℃干燥箱中干燥过夜，取出置于铝箔袋内封口备用。
步骤二：样本垫处理
1)配制磷酸盐缓冲液，并将PH调节至8.5。
2)取一定质量分数的磷酸盐缓冲液：0.01mol/L)、一定质量分数的牛血清白蛋白：0.1％，一定质量分数的表面活性剂Triton X-100：1％、一定质量分数的聚维酮K30：0.05％配置成样本垫处理液。
3)将样本垫处理液均匀分散在玻璃纤维上，37℃干燥箱干燥过夜后取出，再置于铝箔袋内封口备用。
步骤三：结合垫处理
1)将荧光微球溶液的PH调节至8.5，与新型冠状病毒NP蛋白单克隆抗体混匀反应，得到抗体标记物，用复溶液复溶。
2)配制磷酸盐缓冲液，并将PH调节至8.5。
3)取一定质量分数的磷酸盐缓冲液(例如0.01mol/L)、一定质量分数的牛血清白蛋白(例如0.1％)、一定质量分数的蔗糖(例如0.1％)、一定质量分数的proclin300(例如0.01％)配制成结合垫处理液。
4)将处理液均匀分散在玻璃纤维上，37℃干燥箱过夜干燥后取出，再置于铝箔袋内封口备用。
5)将1)中的抗体标记物复溶液用划膜喷金仪以一定的质量分数(例如1uL/cm)喷涂在4)的玻璃纤维上，置于37℃干燥箱过夜干燥后取出，置于铝箔袋封口待用。
步骤四：硝酸纤维素膜(反应膜)划膜
1)配置磷酸盐缓冲液，并将PH调节至8。
2)取一定质量分数的磷酸盐缓冲液(例如0.1mol/L)，将新型冠状病毒NP蛋白单克 隆抗体稀释到1.0mg/ml,作为T线溶液。
3)取一定质量分数的磷酸盐缓冲液(例如0.1mol/L)，将山羊抗鼠多克隆抗体稀释到0.5mg/ml，作为C线溶液。
4)用划膜喷金仪以一定的质量分数(例如0.5uL/cm)进行划膜。
5)将划好的膜置于37℃干燥箱干燥过夜后取出，置于铝箔袋封口待用。
步骤五：组装切割与装袋
1)将步骤一得到的前处理垫，步骤二处理好的样本垫，步骤三得到的结合垫，步骤四处理好的硝酸纤维素膜，吸水纸裁切为适当宽度，依次黏在底板上，每层之间搭牢1-2mm。
2)将粘好的大卡用斩切机斩切成3-6mm宽测试卡，装进卡壳中。
3)将样本采集块固定在卡壳前端。
4)盖上样本采集盖。
5)装入铝箔袋中密封保存。
更进一步的，步骤五中的前处理垫也可以单独黏在底板上裁切，或者无需底板支撑，前处理垫的宽度大于或等于样本垫的宽度。
实施例5另一种检测新型冠状病毒的免疫层析检测装置(荧光法)的制备
与实施例4相同，其区别是使用硬脂酸单乙醇酰胺替代前处理垫制备中的Triton X-100，根据其质量分数的不同分别配置如下三个配方，其它成分与实施例4相同：
配方A：0.01mol/L的磷酸盐缓冲液、质量分数3％的酪蛋白、质量分数0.5％的硬脂酸单乙醇酰胺、质量分数0.01％的proclin 300。
配方B：0.01mol/L的磷酸盐缓冲液、质量分数3％的酪蛋白、质量分数1％的硬脂酸单乙醇酰胺、质量分数0.01％的proclin 300。
配方C：0.01mol/L的磷酸盐缓冲液、质量分数3％的酪蛋白、质量分数1.5％的硬脂酸单乙醇酰胺、质量分数0.01％的proclin 300。
实施例6：实施例2提供的胶体金法制得的检测新型冠状病毒的免疫层析检测装置的验证
将病毒培养物1.51×106TCID50/mL分别稀释成100倍、200倍、400倍、800倍、1600倍、3200倍、6400倍、9600倍作为待测样本，在现有体系中稀释倍数大于6400倍后，判读检测线T线信号时会出现阴性结果。判读检测结果时使用标准比色卡(见图4)进行比对。
如图4所示，若检测线T≥G3，则检测结果为阳性；若检测线T<G3，则检测结果为阴性，质控线始终C≥G8；若质控线C<G8或未显色，即使检测线T显色，测试结果也将无效。
共分三组进行，其中第一组为直接使用无前处理垫唾液检测试剂盒(也不进行样品的裂解)(具体结果见表1)，第二组为先进行样品裂解处理再使用无前处理垫唾液检测试剂盒(具体结果见表2)，第三组为使用实施例2提供的有前处理垫的唾液检测试剂盒进行检测(具体结果见表3)，每一个处理重复20次，使用标准比色卡来判读检测结果(G表示颜色的深浅来判读检测的结果)。第一组，第二组和第三组的测试条结构和试剂相同，除了以下不同：第一组不经过前处理；第二组具有前处理，但是测试条还是传统的无前处理垫的测试条，第三组就是包括前处理垫的测试条。
具体实验过程如下：
1)检测装置在15-30℃复温。
2)将病毒培养物分别稀释成100倍、200倍、400倍、800倍、1600倍、3200倍、6400倍、9600倍。
3)采集唾液样本后分别按照2)中的梯度添加稀释后的的病毒培养物到唾液样本中共得到8份样本，样本的病毒培养物浓度分别为1/9600、1/6400、1/3200、1/1600、1/800、1/400、1/200、1/100，除此之外另有1份样本不添加病毒培养物，其培养物浓度为0。
4)将无前处理垫的现有检测装置及配套的裂解液从密封的铝箔袋中取出；将实施例2提供的有前处理垫的检测装置从密封的铝箔袋中取出，放在干净和水平的平面上。
5)将2)中的每个样本分成三份，其中第二份加入无前处理垫的现有检测装置的裂解液中，第一份和第三份不添加至裂解液中。
6)取60微升未添加裂解液的样本滴加到第一组的检测装置上；取60微升的有裂解液的样本滴加到第二组的检测装置上；将第三组有前处理垫的检测装置的样本采样盖打开，露出样本采样块置入未添加至裂解液中的样本中并静置一定时间(例如1-2分钟)后，盖上样本采样盖，水平方向按压1-2次。
7)室温放置在水平桌面上15～20min后，通过检视窗判读测试结果。
检测结果如表1、表2、表3所示，当稀释倍数相同时，第一组显示结果明显偏弱，难以检测出真实结果(不进行裂解)；第二组和第三组的显色结果一致，色度无差异，因此，采用本发明所述的检测装置进行病毒检测是可行的，在测试条上的样品垫前设置前处理垫，其上包括裂解试剂，可以真正实现无需对样品进行裂解等前处理，直接一步法检测病毒。这样的检测装置不需要额外的前处理步骤，直接滴加到测试条上，减少了步骤，降低了前处理步骤造成对于操作人员可能的二次污染。
表1样本不进行裂解直接加入现有检测装置进行检测的结果
Figure PCTCN2021071116-appb-000001
表2样本进行裂解后加入现有检测装置进行检测的结果
Figure PCTCN2021071116-appb-000002
表3样本直接加入本发明检测装置进行检测的结果
Figure PCTCN2021071116-appb-000003
实施例7实施例3提供的胶体金法制得的另一种检测新型冠状病毒的免疫层析检测装置的验证
将病毒培养物1.51×106TCID50/mL分别稀释成100倍、200倍、400倍、800倍、1600倍、3200倍、6400倍、9600倍作为待测样本，在现有体系中稀释倍数大于6400倍后，判读检测线T线信号时会出现阴性结果。判读检测结果时使用标准比色卡(见图4)进行比对。
如图4所示，若检测线T≥G3，则检测结果为阳性；若检测线T<G3，则检测结果为阴性，质控线始终C≥G8；若质控线C<G8或未显色，即使检测线T显色，测试结果也将无效。
将使用配方1、配方2、配方3制备前处理垫的检测装置分为三组，其中，配方1对应的是第四组(0.5％)，配方2对应的是第五组(1％)，配方3对应的是第六组(1.5％)。采集与实施例6相同的唾液样本进行检测，各组各重复20次，使用标准比色卡来判读检测结果。
具体实验过程的1)至3)与实施例6相同，之后的过程为：
4)将分别依据配方1、配方2、配方3制备前处理垫的检测装置从密封的铝箔袋中取出，放在干净和水平的平面上。
5)将3)中的每个样本分成三份，依次将第四组、第五组、第六组有前处理垫的检测装置的样本采样盖打开，露出样本采样块置入样本中，一个组对应一份样本，并静置与实施例6相同的时间后，盖上样本采样盖，水平方向按压1-2次。
7)室温放置在水平桌面上15～20min后，通过检视窗判读测试结果。
检测结果如表4、表5、表6所示，当把Triton X-100替换为硬脂酸单乙醇酰胺的时候，最低检测阀值在稀释9600倍的时候，仍然具有95％的阳性检测率，与表3相比较，最低检测阀值大大提高。当前处理垫采用配方2制备时，即其中得硬脂酸单乙醇酰胺含量为1％时检测结果的阳性率最高，病毒释放得更完全。另外，发现，稀释9600倍的时候，当硬脂酸单乙醇酰胺含量在1％的时候，阳性检测率是100％。这充分说明，在进行样品前处理垫上处理的裂解试剂中，硬脂酸单乙醇酰胺的加入，对于裂解效果是比较关键的，其质量分数含量优选为1％。
表4样本直接加入实施例3的配方1检测装置进行检测的结果
Figure PCTCN2021071116-appb-000004
表5样本直接加入实施例3的配方2检测装置进行检测的结果
Figure PCTCN2021071116-appb-000005
表6样本直接加入实施例3的配方3检测装置进行检测的结果
Figure PCTCN2021071116-appb-000006
实施例8实施例4提供的荧光法制得的检测新型冠状病毒的免疫层析检测装置的验证
将病毒培养物1.51×106TCID50/mL分别稀释成500倍、1000倍、2000倍、4000倍、5000倍、7500倍、10000倍、12000倍作为待测样本，在现有体系中稀释倍数大于10000倍后，T线信号值表现为与本底值相近，判定为阴性。
共分两组进行，其中第一组为先进行样品裂解处理再使用无前处理垫唾液检测试剂盒，第二组为使用实施例4提供的有前处理垫的唾液检测试剂盒进行检测，重复3次，使用荧光免疫分析仪检测，具体实验过程如下：
1)检测装置在15-30℃复温。
2)将病毒培养物分别稀释成500倍、1000倍、2000倍、4000倍、5000倍、7500倍、10000倍、12000倍，共得到8份样本，样本的病毒培养物浓度分别为1/500、1/1000、1/2000、1/4000、1/5000、1/7500、1/10000、1/12000得唾液样本，除此之外另有1份样本不添加病毒培养物，其培养物浓度为0。
3)采集唾液样本后分别按照2)中的梯度添加稀释后的的病毒培养物。
4)将无前处理垫的现有检测装置及配套的裂解液从密封的铝箔袋中取出，将有前处理垫的检测装置从密封的铝箔袋中取出，放在干净和水平的平面上。
5)将3)中的样本分成两份，其中第一份加入无前处理垫的现有检测装置的裂解液中，第二份不添加至裂解液中。
6)取一定量的有裂解液的样本(如60微升)滴加到第一组的检测装置上；将第二组有前处理垫的检测装置的样本采样盖打开，露出样本采样块置入未添加至裂解液中的样本中并静置一定时间(例如1-2分钟)后盖上样本采样盖，水平方向按压1-2次。
7)室温放置在水平桌面上，15分钟后通过荧光免疫分析仪读取测试结果。
检测结果如表7、表8、表9所示，两组检测结果的质控线C线信号基本稳定，当稀释倍数相同时，检测线T线信号基本相似，取两组数据的T线信号的平均值(Mean T)做线性相关，如表9和图5所示，两组数据的相关性R2＝0.9998，为高度相关数据，因此，采用本发明所述的检测装置进行荧光免疫层析病毒检测是可行的，可以真正实现无需对样品进行裂解等前处理，直接一步法检测病毒。这样不需要额外的前处理步骤，降低了前处理步骤造成对于操作人员可能的二次污染。测试条样品垫前设置的前处理垫上包括裂解试剂，可以获得独立裂解相同的效果。
表7无前处理垫检测装置检测结果
Figure PCTCN2021071116-appb-000007
表8本发明有前处理垫检测装置检测结果
Figure PCTCN2021071116-appb-000008
表9无前处理垫和有前处理垫检测线T线的信号平均值对比
Figure PCTCN2021071116-appb-000009
实施例9实施例5提供的荧光法制得的另一种检测新型冠状病毒的免疫层析检测装置的验证
将病毒培养物1.51×106TCID50/mL分别稀释成500倍、1000倍、2000倍、4000倍、5000倍、7500倍、10000倍、12000倍作为待测样本，在现有体系中稀释倍数大于10000倍后，T线信号值表现为与本底值相近，判定为阴性。
将使用配方A、配方B、配方C制备前处理垫的检测装置分为三组，配方A对应的是第Ⅰ组，配方B对应的是第Ⅱ组，配方C对应的是第Ⅲ组。采集与实施例8相同的唾液样本进行检测，重复3次，使用荧光免疫分析仪检测。
具体实验过程的1)至3)与实施例8相同，之后的过程为：
4)将依据配方A、配方B、配方C制备前处理垫的检测装置从密封的铝箔袋中取出，放在干净和水平的平面上。
5)将3)中的样本分成三份，将第Ⅰ组、第Ⅱ组、第Ⅲ组的检测装置的样本采样盖打开，露出样本采样块置入样本中，一个组对应一份样本，并静置与实施例8相同时间后盖上样本采样盖，水平方向按压1-2次。
7)室温放置在水平桌面上，15分钟后通过荧光免疫分析仪读取测试结果。
检测结果如表10、表11、表12所示，当把Triton X-100替换为硬脂酸单乙醇酰胺的时候，在稀浓度为1/12000时也能检测到信号，相比于实施例8中的检测灵敏度更高，并且当前处理垫采用配方B制备时，即其中得硬脂酸单乙醇酰胺含量为1％时检测结果的阳性率最高，病毒释放得更完全。这充分说明，在进行样品前处理垫上处理的裂解试剂中，硬脂酸单乙醇酰胺的加入，对于裂解效果是比较关键的，其质量分数含量优选为1％。
表10第Ⅰ组检测装置检测结果
Figure PCTCN2021071116-appb-000010
表11第Ⅱ组检测装置检测结果
Figure PCTCN2021071116-appb-000011
表13第Ⅲ组检测装置检测结果
Figure PCTCN2021071116-appb-000012
本发明说明书中提到的所有专利和出版物都表示这些是本领域的公开技术，本发明可以使用。这里所引用的所有专利和出版物都被同样列在参考文献中，跟每一个出版物具体的单独被参考引用一样。这里所述的本发明可以在缺乏任何一种元素或多种元素，一种限制或多种限制的情况下实现，这里这种限制没有特别说明。例如这里每一个实例中术语“包含”，“实质由……组成”和“由……组成”可以用两者之一的其余2个术语代替。这里的所谓的“一个”仅仅表示“一”的意思，而不排除仅仅只是包括一个，也可以表示包括2个以上。这里采用的术语和表达方式所为描述方式，而不受其限制，这里也没有任何意图来指明此书描述的这些术语和解释排除了任何等同的特征，但是可以知道，可以在本发明和权利要求的范围内做任何合适的改变或修改。可以理解，本发明所描述的实施例子都是一些优选的实施例子和特点，任何本领域的一般技术人员都可以根据本发明描述的精髓下做一些更改和变化，这些更改和变化也被认为属于本发明的范围和独立权利要求以及附属权利要求所限制的范围内。

Claims (10)

  1. 一种免疫层析检测装置，其特征在于，包括样本采集部位和试纸条，所述试纸条上包含样品前处理垫，所述样本采集部位中采集的样品可传递至样品前处理垫，样品前处理垫上包括干燥固定的用于病毒样本裂解的前处理试剂。
  2. 如权利要求1所述的检测装置，其特征在于，所述试纸条还包括底板、样本垫、标记垫、反应膜和吸水垫，其中，所述的样品前处理垫、样本垫、标记垫、反应膜和吸水垫依次搭接在一起；所述样本采集部位包括样本采集块和采样槽。
  3. 如权利要求1或2所述的检测装置，其特征在于，所述检测装置还包括卡壳，试纸条装于卡壳内，卡壳前端设有容纳样本采集块的采样槽，样本采集块与试纸条的连接处设有过滤部件。
  4. 如权利要求3所述的检测装置，其特征在于，所述样本采集块为可吸附样品的海绵，样本采集块部分露出采样槽，露出部分套有采样盖，用于挤压样本采集块，使样品通过过滤部件到达样品前处理垫。
  5. 如权利要求4所述的检测装置，其特征在于，所述卡壳包括卡壳盖和卡壳底，卡壳底设有试纸条槽，试纸条槽前端包括前处理垫槽；所述过滤部件为过滤网，所述过滤网位于卡壳盖和/或卡壳底上，过滤网的宽度不大于样品前处理垫的宽度。
  6. 如权利要求5所述的检测装置，其特征在于，样本采集块上设有凹槽，使其固定于卡壳前端的采样槽内，样本采集块与过滤网之间距离1～3mm。
  7. 如权利要求1-6任一项所述的检测装置，其特征在于，所述样品为唾液，所述前处理液试剂为用于唾液样品中的病毒的裂解试剂。
  8. 如权利要求7所述的检测装置，其特征在于，所述裂解试剂包括：0.01mol/L的磷酸盐缓冲液、质量分数3％的酪蛋白、质量分数0.5-1.5％的硬脂酸单乙醇酰胺、质量分数0.01％的proclin 300，其中磷酸盐缓冲液的PH＝7.5-8.5。
  9. 如权利要求2所述的检测装置，其特征在于，所述的标记垫包括胶体金或者荧光标记。
  10. 硬脂酸单乙醇酰胺在用于制备病毒裂解试剂的用途，其特征在于，所述裂解试剂包括：0.01mol/L的磷酸盐缓冲液、质量分数3％的酪蛋白、质量分数0.5-1.5％的硬脂酸单乙醇酰胺、质量分数0.01％的proclin 300，其中磷酸盐缓冲液的PH＝7.5-8.5。
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