CN114720682A - Preparation method of sample pad for immunochromatography detection - Google Patents
Preparation method of sample pad for immunochromatography detection Download PDFInfo
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- CN114720682A CN114720682A CN202210393025.7A CN202210393025A CN114720682A CN 114720682 A CN114720682 A CN 114720682A CN 202210393025 A CN202210393025 A CN 202210393025A CN 114720682 A CN114720682 A CN 114720682A
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- 238000001514 detection method Methods 0.000 title claims abstract description 25
- 238000003317 immunochromatography Methods 0.000 title claims abstract description 13
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 31
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 22
- 210000004369 blood Anatomy 0.000 claims abstract description 21
- 239000008280 blood Substances 0.000 claims abstract description 21
- 238000005303 weighing Methods 0.000 claims abstract description 17
- 229910000162 sodium phosphate Inorganic materials 0.000 claims abstract description 12
- 239000011780 sodium chloride Substances 0.000 claims abstract description 11
- 239000004094 surface-active agent Substances 0.000 claims abstract description 10
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims abstract description 6
- 229910000397 disodium phosphate Inorganic materials 0.000 claims abstract description 6
- 238000009472 formulation Methods 0.000 claims abstract description 6
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims abstract description 6
- 229920002521 macromolecule Polymers 0.000 claims abstract description 6
- 239000000203 mixture Substances 0.000 claims abstract description 6
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims abstract description 6
- 239000012530 fluid Substances 0.000 claims abstract description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims description 35
- 238000000034 method Methods 0.000 claims description 20
- 239000004745 nonwoven fabric Substances 0.000 claims description 18
- 239000002390 adhesive tape Substances 0.000 claims description 16
- 210000002966 serum Anatomy 0.000 claims description 15
- 239000003085 diluting agent Substances 0.000 claims description 6
- 238000007664 blowing Methods 0.000 claims description 5
- 239000000835 fiber Substances 0.000 claims description 3
- 229920000728 polyester Polymers 0.000 claims description 3
- 229920006267 polyester film Polymers 0.000 claims description 3
- 238000003556 assay Methods 0.000 claims 3
- 238000009792 diffusion process Methods 0.000 abstract description 4
- 239000012528 membrane Substances 0.000 description 34
- 239000000020 Nitrocellulose Substances 0.000 description 25
- 229920001220 nitrocellulos Polymers 0.000 description 25
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 16
- 238000010521 absorption reaction Methods 0.000 description 15
- 239000000853 adhesive Substances 0.000 description 7
- 230000001070 adhesive effect Effects 0.000 description 7
- 239000007788 liquid Substances 0.000 description 6
- 229920002366 Tetronic® 1307 Polymers 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 241000709721 Hepatovirus A Species 0.000 description 3
- 241000187479 Mycobacterium tuberculosis Species 0.000 description 3
- 241000202934 Mycoplasma pneumoniae Species 0.000 description 3
- 239000002280 amphoteric surfactant Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000035699 permeability Effects 0.000 description 3
- 238000004445 quantitative analysis Methods 0.000 description 3
- 241001248531 Euchloe <genus> Species 0.000 description 2
- 239000003365 glass fiber Substances 0.000 description 2
- 241001647372 Chlamydia pneumoniae Species 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 239000003945 anionic surfactant Substances 0.000 description 1
- 239000003093 cationic surfactant Substances 0.000 description 1
- 230000009194 climbing Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000005661 hydrophobic surface Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
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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
- G01N33/531—Production of immunochemical test materials
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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Abstract
The invention provides a preparation method of a sample pad used in immunochromatography detection, which is characterized by comprising the following steps: the sample pad is treated with a treatment fluid, the preparation of which comprises the steps of: 1L of sample pad treatment solution was prepared according to the following formulation: preparing 0.2mol/L Na2HPO4: weighing 71.6g Na2HPO4‑12H2O, dissolving in 1000ml of water to prepare 0.2mol/L NaH2PO4: weighing 31.2g of NaH2PO4‑2H2O, then 19ml of 0.2mol/L Na is taken2HPO481ml of 0.2mol/L NaH2PO40.2mol/LPB is prepared, and then 100ml of 0.2mol/L PB is taken and diluted to 1000ml by adding water to obtain 0.Weighing 9g of NaCl in a 02mol/LPB solution, adding the NaCl into the 0.02mol/LPB solution to obtain 0.02mol/LPBS, sequentially adding 0.5g of anti-RBC, 5g of surfactant S9 and 4g of macromolecular substance PVP10, and measuring 0.5mL of Proclin-300. The sample can be uniformly diffused on the sample cushion layer, the problem of nonuniform sample application and diffusion of the whole blood sample is solved, and the color development of the detection line (T) area is uniform.
Description
Technical Field
The invention relates to a sample pad, in particular to a preparation method of the sample pad used for immunochromatography detection.
Background
The Immunochromatography (ICA) is a novel immunoassay method appearing in the early 80 th century, and the principle of the Immunochromatography (ICA) is that a specific antigen or antibody is fixed on a membrane in a strip shape, a colloidal gold labeled reagent (antibody or monoclonal antibody) is adsorbed on a binding pad, after a sample to be detected is added to a sample pad at one end of a test strip, the sample moves forward by capillary action, the colloidal gold labeled reagent on the binding pad is dissolved and reacts with each other, and when the sample moves to a region of the fixed antigen or antibody, the conjugate of the sample and the gold labeled reagent is specifically bound with the sample and is retained, and is gathered on a detection strip, and a color development result can be observed by naked eyes. The method is developed into a diagnostic test strip, and is very convenient to use.
The detection principle in immunochromatography includes a double-antigen sandwich method, a double-antibody sandwich method, a competition method, an indirect method and the like, wherein the indirect method is mainly used for detecting antibodies in serum, purified or recombinant antigens are fixed on an NC membrane, most of the markers are proteins, the method requires excessive colloidal gold, generally, a sample needs to be diluted before being added or is added with a diluent after being added with a very small amount,
the indirect method principle, the application of sample method is the membrane application of sample method, use pipettor to get 1.5 mul serum/plasma or 2 mul whole blood sample and add perpendicularly in sample application of sample department, and the application of sample must be in order to touch NC membrane gently in the liquid ware rifle head when the application of sample so that the application of sample smoothly. If the disposable plastic sample adding ring is used, the ring body needs to be completely immersed into a sample, then the ring body vertically lightly touches the membrane surface of the reagent sample adding position to infiltrate and absorb the sample, and the NC membrane surface of the sample adding position has a wetting trace, so that the successful sample adding is prompted.
The invention has the advantages that: the invention greatly reduces the sample dosage, realizes the peripheral blood detection and is convenient for the patient to take blood. Serum, plasma and whole blood samples can be detected, and the requirements of different people and detection environments are met. At present, the sample adding method is applied to products such as mycobacterium tuberculosis IgG antibody, mycoplasma pneumoniae IgG antibody, chlamydia pneumoniae IgG antibody, hepatitis A virus IgG antibody and the like.
However, the sample adding method has the problems that the phenomenon of uneven diffusion exists when the whole blood sample is added at the NC membrane, the phenomenon that T lines are uneven or are half-way appears in the detection line (T) area in the sample climbing process, and the result interpretation is influenced.
Disclosure of Invention
The invention aims to overcome the defects of the traditional technology and provides a preparation method of a sample pad for immunochromatography detection.
The aim of the invention is achieved by the following technical measures: a method for preparing a sample pad used in immunochromatography detection is characterized in that: the sample pad is treated by a treatment fluid, and the preparation of the treatment fluid comprises the following steps:
1L of sample pad treatment solution was prepared according to the following formulation: preparing 0.2mol/L Na2HPO4: weighing 71.6g Na2HPO4-12H2O, dissolving in 1000ml of water to prepare 0.2mol/L NaH2PO4: weighing 31.2g of NaH2PO4-2H2O, then 19ml of 0.2mol/L Na is taken2HPO481ml of 0.2mol/L NaH2PO4Preparing 0.2mol/LPB, then taking 100ml of 0.2mol/L PB, adding water to dilute to 1000ml to obtain 0.02mol/LPB solution, weighing 9g of NaCl, adding the NaCl into the 0.02mol/LPB solution to obtain 0.02mol/LPBS, and then sequentially adding 0.5g of anti-RBC, 5g of surfactant S9 and 4g of macromolecular substancesPVP10, 0.5mL Proclin-300 was measured.
As a preferred option, the sample pad is treated with a treatment solution that is configured to: each 30cm × 25cm sample pad was uniformly coated with 50mL of the treatment solution, dried in an electrothermal blowing dry oven at 37 ℃ for 12 hours, and stored for further use.
Preferably, the processed special sample pad is cut into 1 × 10mm, the sample pad is attached to the position of the sample application hole of the reagent strip by using a double-sided adhesive tape and moved down by 1 × 10mm, and then the sample pad is placed on the attached double-sided adhesive tape, so that the sample pad is just positioned at the sample application position.
As a preferred scheme, the sample adding steps are as follows:
taking 1.5 mu l of serum/plasma or 2 mu l of whole blood sample by using a pipettor, or taking 1.5 mu l of serum/plasma or 2 mu l of whole blood sample by using a sample adding ring;
moving a pipette tip or a disposable plastic sample adding ring, and vertically adding the pipette tip or the disposable plastic sample adding ring to a sample adding position;
controlling a pipette tip or a disposable plastic sample adding ring to be arranged at a sample adding hole;
immediately below the S well, 2 drops of diluent (about 80-100. mu.l) were added dropwise, and the results were observed for 15-20 minutes, after 20 minutes the results were invalid.
Preferably, the sample pad is made of non-woven fabric, polyester fiber or polyester film.
Due to the adoption of the technical scheme, compared with the prior art, the invention has the advantages that: the special processed sample pad added at the sample adding position replaces a nitrocellulose membrane to bear serum, plasma and whole blood samples, can enable the samples to be uniformly diffused on the sample pad without uniformly smearing the samples, can be uniformly diffused without damaging the nitrocellulose membrane, has the problem of non-uniformity in diffusion uniformity after the samples are added due to the fact that an NC membrane has certain hydrophilicity at the position of the membrane sample adding position, selects non-woven fabrics as the sample pad to be processed and used, the sample pad is an important component part in an immunity rapid test reagent strip and mainly comprises glass fibers, non-woven fabrics and the like, generally uses chemical reagents to modify the sample pad in order to uniformly distribute the samples, plays a role in pre-filtering, and on the other hand, because glass fiber processing is used for smearing, the condition of non-uniform smearing exists, therefore, the non-woven fabrics are selected as the sample pad to be used, the liquid can be uniformly diffused into the non-woven fabric without being smeared;
in the selection of the treatment liquid, surfactant S9 (Tetronic 1307) with good dispersibility and permeability is selected, and the Tetronic 1307 has good interfacial activity, the advantages of both anionic and cationic surfactants, and good emulsifying capacity, dispersing capacity, antistatic effect and the like. Compared with other surfactants, the amphoteric surfactant is not easily affected by inorganic electrolyte and has no cloud point phenomenon, and no matter the amphoteric surfactant is adsorbed to a positive charge interface or a negative charge interface, a hydrophobic surface cannot be formed, and the characteristics enable the amphoteric surfactant to have wider application potential in many fields than a nonionic surfactant, so that S9 (Tetronic 1307) is added into the non-woven fabric treatment solution in order to enable the sample to be more uniformly diffused at a sample loading position;
in addition, PVP10 is added into the treatment solution, the efficacy is similar to that of a surfactant, the surface tension of water can be reduced by high molecular weight PVP10 at a proper concentration, the water solubility of certain active substances which are basically insoluble in water can be increased, the dispersion effect is realized, and the colored substances, the suspension and the emulsion in the solution can be uniformly dispersed and kept stable; in addition, it can adsorb on many interfaces and reduce the interfacial surface tension to some extent.
The invention adopts the non-woven fabric sample pad material with good permeability and diffusivity to replace the nitrocellulose membrane to bear serum, plasma and whole blood samples, can uniformly and effectively absorb liquid, and ensures that the liquid is uniformly diffused on the sample pad without uniformly smearing the sample, and can be uniformly diffused without damaging the nitrocellulose membrane.
The surfactant S9 (Tetronic 1307) with better dispersibility and permeability and the macromolecular substance PVP10 which can reduce the surface tension and increase the water solubility are added into the processing liquid of the sample pad, so that the sample can be uniformly diffused on the sample pad layer, the problem of nonuniform sample application and diffusion of the whole blood sample is solved, and the color development of a detection line (T) area is uniform.
The invention is further described with reference to the following figures and detailed description.
Drawings
Fig. 1 is a schematic structural view of embodiment 1 of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the description of the present invention, it is to be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", and the like, indicate orientations and positional relationships based on those shown in the drawings, and are used only for convenience of description and simplicity of description, and do not indicate or imply that the equipment or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be considered as limiting the present invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.
Example 1: a mycobacterium tuberculosis IgG (TB-IgG) antibody detection test strip is shown in figure 1 and comprises a bottom plate 1, wherein the bottom plate 1 is a PVC plate, a cushion pad 2, a gold-labeled cushion layer 3, a nitrocellulose membrane 4, a sample cushion layer 5 and a water absorption layer 6 which are sequentially overlapped are adhered on the bottom plate 1, the water absorption layer 6 is water absorption paper, one end of the cushion pad 2 presses one end of the gold-labeled cushion layer 3 for 0.5-1mm, the other end of the gold-labeled cushion layer 3 presses one end of the nitrocellulose membrane 4 for 0.5-1mm, one end of the water absorption layer 6 presses the other end of the nitrocellulose membrane 4 for 0.5-1mm, the prepared nitrocellulose membrane 4 is adhered at the middle position of the bottom plate 1, and the water absorption layer 6 is adhered above the position of the bottom plate 1 where the nitrocellulose membrane 4 is fixed; paste little white subsides 7 above the gold mark bed course 3 and 4 handing-over parts of nitrocellulose membrane and press 40.5-1mm of nitrocellulose membrane, fix sample bed course 5 above little white subsides 7, move down 1 x 10mm department in reagent strip application of sample hole position with double faced adhesive tape subsides, paste the sample bed course 5 that the width is 1 x 10mm on double faced adhesive, with the parallel and level of double faced adhesive tape lower extreme for the sample pad is located the application of sample position just. And (3) flattening the product obtained in the step, and cutting the product into test strips with the width of 4.0 mm.
The sample cushion layer is made of non-woven fabric, polyester fiber or polyester film.
The sample pad treatment solution mainly comprises 0.02MPBS, 0.5mg/ml anti-RBC, 0.5% S9, 0.4% PVP10 and 0.05% Proclin-300, after dissolution, the non-woven fabric sample pad is subjected to saturation treatment, and after drying, the non-woven fabric sample pad is assembled with the reagent strip.
As an optimized solution, the preparation of the special sample pad treatment solution comprises the following steps:
1L of sample pad treatment solution was prepared according to the following formulation: preparing 0.2mol/L Na2HPO4: weighing 71.6g Na2HPO4-12H2O dissolved in 1000ml of water to prepare 0.2mol/L NaH2PO4: 31.2gNaH was weighed out2PO4-2H2O, then 19ml of 0.2mol/L Na is taken2HPO481ml of 0.2mol/L NaH2PO4Preparing 0.2mol/LPB, then taking 100mL of 0.2mol/L PB, adding water to dilute to 1000mL to obtain 0.02mol/LPB solution, weighing 9g of NaCl, adding the NaCl into the 0.02mol/LPB solution to obtain 0.02mol/LPBS, then sequentially adding 0.5g of anti-RBC, 5g of surfactant S9, 4g of macromolecular substance PVP10, and measuring 0.5mL of Proclin-300.
Treating the sample pad with the prepared sample pad treatment solution: each 30cm × 25cm sample pad was uniformly coated with 50mL of the treatment solution, dried in an electrothermal blowing dry oven at 37 ℃ for 12 hours, and stored for later use.
And assembling the special sample pad with the reagent strip, cutting the processed special sample pad into 1 x 10mm, pasting the special sample pad on the position of a sample adding hole of the reagent strip by using a double-sided adhesive tape, moving the position downwards by 1 x 10mm, and then placing the sample pad on the pasted double-sided adhesive tape to ensure that the sample pad is just positioned at the sample adding position.
The sample adding steps are as follows:
(1) taking 1.5 mu l of serum/plasma or 2 mu l of whole blood sample by using a pipettor, or taking 1.5 mu l of serum/plasma or 2 mu l of whole blood sample by using a sample adding ring;
(2) moving a pipette tip or a disposable plastic sample adding ring, and vertically adding the pipette tip or the disposable plastic sample adding ring to a sample adding position;
(3) the pipette tip or the disposable plastic sample adding ring is controlled to be arranged at the position of the sample adding hole, and uniform smearing is not needed;
(4) immediately below the S well, 2 drops of diluent (about 80-100. mu.l) were added dropwise, and the results were observed for 15-20 minutes, after 20 minutes the results were invalid.
The reagent (hereinafter referred to as reagent 1) for detecting antibody of mycobacterium tuberculosis IgG (TB-IgG) assembled as above, and the reagent (hereinafter referred to as reagent 2) assembled with the non-woven fabric sample pad are normally operated according to the sample adding step, 523 samples of whole blood are detected by comparison, and in 106 samples of detected positive samples, the color development uniformity of two reagent detection lines (T) is compared; the data of the color development of the detection line (T) after completion of the detection are analyzed as shown in Table 1:
TABLE 1 quantitative analysis of T-line color development uniformity in Positive samples
Example 2: a mycoplasma pneumoniae IgG (MP-IgG) antibody detection test strip comprises a base plate, wherein the base plate is a PVC plate, a cushion pad, a gold-labeled cushion layer, a nitrocellulose membrane, a sample cushion layer and a water absorption layer which are sequentially overlapped are stuck on the base plate, the water absorption layer is water absorption paper, one end of the cushion pad presses one end of the gold-labeled cushion layer by 0.5-1mm, the other end of the gold-labeled cushion layer presses one end of the nitrocellulose membrane by 0.5-1mm, one end of the water absorption layer presses the other end of the nitrocellulose membrane by 0.5-1mm, the prepared nitrocellulose membrane is stuck at the middle position of the base plate, and the water absorption layer is stuck above the position where the nitrocellulose membrane is fixed on the base plate; pasting a small white adhesive nitrocellulose membrane 0.5-1mm above the joint of the gold-labeled cushion layer and the nitrocellulose membrane, fixing the sample cushion layer above the small white adhesive, pasting a double-sided adhesive tape at the position of a sample adding hole of the reagent strip, moving the position by 1 x 10mm downwards, pasting the sample cushion layer with the width of 1 x 10mm on the double-sided adhesive, and aligning with the lower end of the double-sided adhesive tape, so that the sample cushion is just positioned at the sample adding position. And flattening the product obtained in the step, and cutting the product into test strips with the width of 4.0 mm.
The special sample pad treatment solution mainly comprises 0.02MPBS, 0.5mg/ml anti-RBC, 0.7% S9, 0.5% PVP10 and 0.07% Proclin-300, after dissolution, the non-woven fabric sample pad is subjected to saturation treatment, and after drying, the non-woven fabric sample pad is assembled with the reagent strip.
As an optimized solution, the preparation of the special sample pad treatment solution comprises the following steps:
1L of sample pad treatment solution was prepared according to the following formulation: preparing 0.2mol/L Na2HPO4: weighing 71.6g Na2HPO4-12H2O, dissolving in 1000ml of water to prepare 0.2mol/L NaH2PO4: weighing 31.2g of NaH2PO4-2H2O, then 19ml of 0.2mol/L Na is taken2HPO481ml of 0.2mol/L NaH2PO4Preparing 0.2mol/LPB, then taking 100mL of 0.2mol/L PB, adding water to dilute to 1000mL to obtain 0.02mol/LPB solution, weighing 9g of NaCl, adding the NaCl into the 0.02mol/LPB solution to obtain 0.02mol/LPBS, then sequentially adding 0.5g of anti-RBC, 5g of surfactant S9, 4g of macromolecular substance PVP10, and measuring 0.5mL of Proclin-300.
Treating the sample pad with the prepared sample pad treatment solution: each 30cm × 25cm sample pad was uniformly coated with 50mL of the treatment solution, dried in an electrothermal blowing dry oven at 37 ℃ for 12 hours, and stored for further use.
And assembling the special sample pad with the reagent strip, cutting the processed special sample pad into 1 x 10mm, pasting the special sample pad on the position of a sample adding hole of the reagent strip by using a double-sided adhesive tape, moving the position downwards by 1 x 10mm, and then placing the sample pad on the pasted double-sided adhesive tape to ensure that the sample pad is just positioned at the sample adding position.
The sample adding steps are as follows:
(1) taking 1.5 mu l of serum/plasma or 2 mu l of whole blood sample by using a pipettor, or taking 1.5 mu l of serum/plasma or 2 mu l of whole blood sample by using a sample adding ring;
(2) moving a pipette tip or a disposable plastic sample adding ring, and vertically adding the pipette tip or the disposable plastic sample adding ring to a sample adding position;
(3) the pipette tip or the disposable plastic sample adding ring is controlled to be arranged at the position of the sample adding hole, and uniform smearing is not needed;
(4) immediately below the S well, 2 drops of diluent (about 80-100. mu.l) were added dropwise, and the results were observed for 15-20 minutes, after 20 minutes the results were invalid.
The reagent (hereinafter referred to as reagent 3) for detecting IgG antibody against Mycoplasma pneumoniae assembled as above and the reagent (hereinafter referred to as reagent 4) assembled without nonwoven fabric sample pad were subjected to the normal operation according to the sample application procedure, and then 676 samples of whole blood were subjected to the comparative detection, and the uniformity of color development of the two reagent detection lines (T) was compared in 203 samples of positive samples detected; the data of the color development of the detection line (T) after completion of the detection are analyzed as shown in Table 2:
TABLE 2 quantitative analysis of T-line color development uniformity in positive samples
Example 3:
a hepatitis A virus IgG (HAV-IgG) antibody detection test strip comprises a bottom plate, wherein the bottom plate is a PVC plate, a cushion pad, a gold-labeled cushion layer, a nitrocellulose membrane, a sample cushion layer and a water absorption layer which are sequentially overlapped are stuck on the bottom plate, the water absorption layer is water absorption paper, one end of the cushion pad presses one end of the gold-labeled cushion layer by 0.5-1mm, the other end of the gold-labeled cushion layer presses one end of the nitrocellulose membrane by 0.5-1mm, one end of the water absorption layer presses the other end of the nitrocellulose membrane by 0.5-1mm, the prepared nitrocellulose membrane is stuck at the middle position of the bottom plate, and the water absorption layer is stuck above the position where the nitrocellulose membrane is fixed on the bottom plate; pasting a small white adhesive nitrocellulose membrane 0.5-1mm above the joint of the gold-labeled cushion layer and the nitrocellulose membrane, fixing the sample cushion layer above the small white adhesive, pasting a double-sided adhesive tape at the position of a sample adding hole of the reagent strip, moving the position by 1 x 10mm downwards, pasting the sample cushion layer with the width of 1 x 10mm on the double-sided adhesive, and aligning with the lower end of the double-sided adhesive tape, so that the sample cushion is just positioned at the sample adding position. And (3) flattening the product obtained in the step, and cutting the product into test strips with the width of 4.0 mm.
The special sample pad treatment solution mainly comprises 0.02MPBS, 0.5mg/ml anti-RBC, 0.6% S9, 0.6% PVP10 and 0.07% Proclin-300, after dissolution, the non-woven fabric sample pad is subjected to saturation treatment, and after drying, the non-woven fabric sample pad is assembled with the reagent strip.
As an optimized solution, the preparation of the special sample pad treatment solution comprises the following steps:
1L of sample pad treatment solution was prepared according to the following formulation: preparing 0.2mol/L Na2HPO4: weighing 71.6g Na2HPO4-12H2O, dissolving in 1000ml of water to prepare 0.2mol/L NaH2PO4: weighing 31.2g of NaH2PO4-2H2O, then 19ml of 0.2mol/L Na is taken2HPO481ml of 0.2mol/L NaH2PO4Preparing 0.2mol/LPB, then taking 100mL of 0.2mol/L PB, adding water to dilute to 1000mL to obtain 0.02mol/LPB solution, weighing 9g of NaCl, adding the NaCl into the 0.02mol/LPB solution to obtain 0.02mol/LPBS, then sequentially adding 0.5g of anti-RBC, 5g of surfactant S9, 4g of macromolecular substance PVP10, and measuring 0.5mL of Proclin-300.
Treating the sample pad with the prepared sample pad treatment solution: each 30cm × 25cm sample pad was uniformly coated with 50mL of the treatment solution, dried in an electrothermal blowing dry oven at 37 ℃ for 12 hours, and stored for further use.
And assembling the special sample pad with the reagent strip, cutting the processed special sample pad into 1 x 10mm, pasting the special sample pad on the position of a sample adding hole of the reagent strip by using a double-sided adhesive tape, moving the position downwards by 1 x 10mm, and then placing the sample pad on the pasted double-sided adhesive tape to ensure that the sample pad is just positioned at the sample adding position.
The sample adding steps are as follows:
(1) taking 1.5 mu l of serum/plasma or 2 mu l of whole blood sample by using a pipettor, or taking 1.5 mu l of serum/plasma or 2 mu l of whole blood sample by using a sample adding ring;
(2) moving a pipette tip or a disposable plastic sample adding ring, and vertically adding the pipette tip or the disposable plastic sample adding ring to a sample adding position;
(3) the pipette tip or the disposable plastic sample adding ring is controlled to be arranged at the position of the sample adding hole, and uniform smearing is not needed;
(4) immediately below the S-well, 2 drops of diluent (about 80-100. mu.l) were added dropwise, and the results were observed for 15-20 minutes, after 20 minutes, the results were invalid.
The reagent (hereinafter referred to as reagent 5) for detecting hepatitis A virus IgG (HAV-IgG) antibody assembled as described above was compared with a reagent (hereinafter referred to as reagent 6) assembled without a nonwoven fabric sample pad to detect 612 whole blood samples, and the uniformity of color development of the two reagent detection lines (T) was compared in 401 positive samples detected; the data of the color development of the test line (T) after completion of the test are analyzed as shown in Table 3:
TABLE 3 quantitative analysis of T-line color development uniformity in positive samples
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (5)
1. A method for preparing a sample pad used in immunochromatography detection is characterized in that: the sample pad is treated by a treatment fluid, the preparation of which comprises the steps of:
1L of sample pad treatment solution was prepared according to the following formulation: preparing 0.2mol/L Na2HPO4: weighing 71.6g Na2HPO4-12H2O, dissolving in 1000ml of water to prepare 0.2mol/L NaH2PO4: weighing 31.2g of NaH2PO4-2H2O, then 19ml of 0.2mol/L Na is taken2HPO481ml of 0.2mol/L NaH2PO4Preparing 0.2mol/LPB, then taking 100mL of 0.2mol/L PB, adding water to dilute to 1000mL to obtain 0.02mol/LPB solution, weighing 9g of NaCl, adding into the 0.02mol/LPB solution to obtain 0.02mol/LPBS, then sequentially adding 0.5g of anti-RBC, 5g of surfactant S9, 4g of macromolecular substance PVP10, and measuring 0.5mL of Proclin-300.
2. The method of claim 1, wherein the step of preparing the sample pad for use in immunochromatographic assay comprises: treating the sample pad with the prepared treatment solution: each 30cm × 25cm sample pad was uniformly coated with 50mL of the treatment solution, dried in an electrothermal blowing dry oven at 37 ℃ for 12 hours, and stored for further use.
3. The method of claim 2, wherein the step of preparing the sample pad for immunochromatography test comprises: cutting the processed special sample pad into 1 x 10mm, pasting the special sample pad on the position of a sample adding hole of the reagent strip by using a double-sided adhesive tape, moving the position downwards by 1 x 10mm, and then placing the sample pad on the pasted double-sided adhesive tape so that the sample pad is just positioned at the sample adding position.
4. The method for preparing a sample pad for immunochromatographic assay according to claim 3, wherein: the sample adding steps are as follows:
taking 1.5 mu l of serum/plasma or 2 mu l of whole blood sample by using a pipettor, or taking 1.5 mu l of serum/plasma or 2 mu l of whole blood sample by using a sample adding ring;
moving a pipette tip or a disposable plastic sample adding ring, and vertically adding the pipette tip or the disposable plastic sample adding ring to a sample adding position;
controlling a pipette tip or a disposable plastic sample adding ring to be arranged at a sample adding hole;
immediately dropping 2 drops of diluent into the S hole on the lower part, observing the result after 15-20 minutes, and after 20 minutes, the result is invalid.
5. The method for preparing a sample pad for use in immunochromatographic assay according to any one of claims 1 to 4, wherein: the sample pad is made of non-woven fabrics, polyester fibers or polyester films.
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