CN106442079B - Blood filtration sample pad and preparation method thereof - Google Patents
Blood filtration sample pad and preparation method thereof Download PDFInfo
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- CN106442079B CN106442079B CN201610795707.5A CN201610795707A CN106442079B CN 106442079 B CN106442079 B CN 106442079B CN 201610795707 A CN201610795707 A CN 201610795707A CN 106442079 B CN106442079 B CN 106442079B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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Abstract
A blood filtration sample pad is prepared by preparing a certain proportion of human erythrocyte antibody, trihydroxymethyl aminomethane, casein, polyvinylpyrrolidone, surfactant and the like into treatment solutions according to different product performances in different orders, and adjusting the pH of the prepared solution to form a final treatment solution; and uniformly treating the prepared treatment solution on glass fiber, and drying in an oven at 37 ℃ for 8-24 hours. The blood filtering sample pad prepared by the method can well separate the whole blood sample under the condition of not influencing sample running, and the background of the rapid diagnostic reagent after sample adding is clean and has no phenomena of blood residue trace, no floating, no line whitening, no track and the like.
Description
Technical Field
The invention relates to the field of medicine inspection, in particular to a whole blood separation membrane for whole blood inspection and a preparation method thereof.
Background
The blood filtering sample pad at the present stage uses a whole blood separation membrane which is also called a blood filtering membrane or a plasma separation membrane as a main material, can rapidly separate plasma in whole blood without hemolysis, so that a client who uses a product at the end does not need to centrifuge blood into serum, further saves detection time and increases efficiency, and can directly use peripheral blood or fresh blood. The whole blood separated by the blood filtering membrane intercepts red blood cells and white blood cells in the whole blood by a physical separation mode.
The whole blood separation membrane is added on the test reagent strip, so that an additional device is not needed, and the whole blood separation membrane is convenient to carry and use; the speed of separating the plasma is high; by selecting a suitable blood filtration membrane, the hemolysis potential is low. However, the existing blood filtration membranes have certain defects, such as: 1. when the reagent strip is used for detecting a whole blood sample, the running speed and the detection result of the detected sample are often influenced, and the phenomena of blood residual traces, line whitening and the like occur. 2. In the prior art, aiming at different packaging forms of final products, the length requirements of films are different, and targeted debugging is needed; 3. when the blood filtering membrane is used, a layer of membrane sealing adhesive tape needs to be added, so that the thickness of the reagent strip is increased, and the production operation is not facilitated; 4. in the process of adhering the blood filtering membrane, the front and back sides need to be carefully operated, and operation errors are easy to occur in mass production.
Disclosure of Invention
The invention aims to solve the technical problem of providing a sample pad which is simpler to operate and has better blood filtration effect and a preparation method thereof.
A blood filtration sample pad comprising glass fibers and a treatment solution for the glass fibers, the treatment solution comprising purified water, anti-RBCs, a surfactant, a phosphate buffer, and tris, casein, and polyvinylpyrrolidone, wherein the anti-RBCs comprise murine anti-RBCs or ovine anti-RBCs.
The preparation method of the treatment solution of the blood filtration sample pad is as follows:
1) taking purified water with the amount of 0.9 required by the treatment solution, sequentially adding 0.04-0.06M/L of trihydroxymethyl aminomethane, 0.15-0.25 mM of casein and 0.15-0.35 mM of polyvinylpyrrolidone into the purified water, adding the next component after the former component is completely dissolved by stirring, and stirring until the next component is completely dissolved;
2) sequentially adding 0.005-0.015M of sodium carbonate, 0.6-1.1% of surfactant of a solution to be treated and 3-5% of mouse-derived anti-RBC of the solution to be treated into the prepared solution, stirring and completely dissolving the former added component, adding the next component, and stirring and completely dissolving;
3) adjusting the pH value of the solution to 8.0 +/-0.1;
4) the prepared solution was made to volume with purified water to the required solution volume and the pH was re-measured at 8.0 ± 0.1.
Yet another method of preparing the treatment solution for the hemofiltration sample pad is as follows:
1) taking purified water with the amount of 0.9 required by the treatment solution, sequentially adding 0.04-0.06M of tris (hydroxymethyl) aminomethane, 0.15-0.25 mM of casein and 0.15-0.35 mM of polyvinylpyrrolidone into the purified water, stirring the former component to be completely dissolved, adding the next component, and stirring to be completely dissolved;
2) adding the following components in the solution prepared in the step 1) in sequence: 0.01-0.015M of Na2CO30.2-0.6% of surfactant in the solution to be treated, stirring the former component to be completely dissolved, adding the next component, and stirring until the next component is completely dissolved;
3) adjusting the pH value of the solution to 8.0 +/-0.1;
4) adding the following components into the solution with the adjusted pH value: 3-5% of mouse-derived anti-RBC in the required treatment solution is stirred until the solution is clear;
5) the solution was made up to the desired treatment solution volume with purified water and the pH was re-measured at 8.0. + -. 0.1.
Yet another method of preparing the treatment solution for the hemofiltration sample pad is as follows:
1) taking purified water with the amount of 0.7 required by the treatment solution, sequentially adding 0.04-0.06M of tris (hydroxymethyl) aminomethane, 0.1-0.3 mM of polyvinylpyrrolidone, 0.02-0.1% of surfactant, 0.15-0.25 mM of casein, 0.02-0.04M of ethylenediamine tetraacetic acid, 8-12% of protein stabilizer of the required treatment solution and 2-5% of ovine anti-RBC of the required treatment solution into the purified water, stirring the former component to be completely dissolved, adding the next component, and stirring to be completely dissolved;
2) adjusting the pH value of the solution to 8.0 +/-0.1;
3) the solution with the adjusted pH value is subjected to constant volume to the required volume of the solution by using purified water, and the pH value is repeatedly measured to be 8.0 +/-0.1;
yet another method of preparing the treatment solution for the hemofiltration sample pad is as follows:
1) taking purified water with the amount of 0.8 of the treatment solution, sequentially adding 0.08-0.12M of trihydroxymethyl aminomethane, 0.3-0.5 mM of polyvinylpyrrolidone, 0.02-0.04M of ethylene diamine tetraacetic acid, 8-12% of protein stabilizer, 0.2-0.6% of surfactant and 0.15-0.25 mM of casein into the purified water, stirring the former component to be completely dissolved, adding the next component, and stirring to be completely dissolved;
2) adjusting the pH value of the solution to 8.0 +/-0.1;
3) adding the following components into the solution with the adjusted pH value: 3-5% of mouse-derived anti-RBC in the required treatment solution is stirred until the solution is clear;
4) the solution is metered to the volume of the required treatment solution, and the pH value is measured again to be 8.0 +/-0.1.
Preferably, the solution for adjusting pH is any one of hydrochloric acid or sodium hydroxide solution.
The preparation method of the blood filtering sample pad comprises the following steps:
1) preparing the treatment solution;
2) uniformly treating the prepared treatment solution on glass fibers;
3) and (3) drying the glass fiber treated by the treatment solution for 8-24 hours at 37 ℃.
The invention has the beneficial effects that:
1. the component proportion and the processing method of the processing solution of the blood filtering sample pad can enable the blood filtering sample pad to well separate a whole blood sample under the condition of not influencing a sample running plate, and a large number of experimental results and evidence collection show that the rapid diagnosis reagent strip after the blood filtering sample pad is applied with sample has a clean background and does not have the phenomena of blood residual trace, Flooding, whitening of a wireless strip, a track and the like.
2. The preparation method of the processing solution of the sample pad can improve the uniformity of the sample, regulate the release speed of the sample, improve the dissolving capacity of the detected substance in the sample pad, eliminate false positive to a certain extent and enable the immune protein conjugate on the detection line to be more stable.
3. The blood filtration sample pad can not be adjusted along with the change of the width of the reagent strip; the mold sealing adhesive tape is not needed, and the production process is simplified; the production operation is convenient, the front and the back of the sample pad do not need to be distinguished specially, and the production efficiency is improved; and under the condition of unchanged product quality, the production cost is reduced, and the enterprise development is promoted.
Detailed Description
The hemofilter sample pad is a glass fiber pad treated with a buffered saline solution. The processing solution of the blood filtration sample pad contains human erythrocyte antibodies (hereinafter, abbreviated as anti-RBC), Tris (Tris), Casein (hereinafter, abbreviated as Casein), polyvinylpyrrolidone (hereinafter, abbreviated as PVP), a surfactant, ethylenediaminetetraacetic acid, and a protein stabilizer in a certain proportion. Different anti-RBC and other auxiliary solutes are added according to different product performances to achieve the best detection effect.
The preparation steps of the sample pad are as follows:
1. preparing raw materials of each component into a solution according to corresponding proportion and sequence according to different product performances, and adjusting the pH value of the prepared solution to form an optimal buffer system suitable for antigen-antibody reaction;
2. calculating the required amount of the solution according to the liquid absorption coefficient of the corresponding glass fiber, and uniformly treating the prepared treatment solution on the glass fiber;
3. and (3) drying the glass fiber treated by the treatment solution in a 37 ℃ oven or drying room for 8-24 hours.
Example 1: blood filtration sample pad treatment of human immunodeficiency virus (HIV 1/2) antibody detection reagent (latex method) product
The preparation steps of the blood filtration sample pad are as follows:
1) preparing purified water with the amount of 0.9 required by the solution, sequentially adding 0.04-0.06M of Tris, 0.15-0.25 mM of Casein and 0.15-0.35 mM of PVP, stirring the former component to be completely dissolved, adding the next component, and stirring to be completely dissolved;
2) adding in sequence to the solution prepared in step 1): 0.01 to 0.015M sodium carbonate (hereinafter abbreviated as Na)2CO3) 0.6-1.1% of surfactant and 3-5% of murine anti-RBC in the solution to be treated, stirring the former component for complete dissolution, adding the next component for stirring for complete dissolution, and clarifying the solution;
3) adjusting the pH value to 8.0 +/-0.1 by using hydrochloric acid or sodium hydroxide solution;
4) fixing the prepared solution to the required volume by using purified water, and repeatedly measuring the pH value to be 8.0 +/-0.1;
5) and uniformly treating the prepared treatment solution on glass fiber, and drying for 8-24 hours in a drying oven or drying room at 37 ℃.
Example 2 hepatitis B Virus surface antigen detection reagent (colloidal gold method) product sample pad treatment
The preparation steps of the sample pad are as follows:
1) taking purified water with the amount of 0.7 required by a treatment solution, sequentially adding 0.04-0.06M of Tris, 0.1-0.3 mM of PVP, 0.02-0.1% of surfactant of the treatment solution, 0.15-0.25 mM of Casein, 0.02-0.04M of ethylenediamine tetraacetic acid, 8-12% of protein stabilizer of the treatment solution and 2-5% of sheep-derived anti-RBC of the treatment solution into the purified water, stirring the former component to be completely dissolved, adding the next component, stirring to be completely dissolved, and clarifying the solution;
2) adjusting the pH value of the solution to 8.0 +/-0.1 by using hydrochloric acid or sodium hydroxide solution;
3) fixing the volume of the solution to the required volume of the solution by using purified water, and repeatedly measuring the pH value to be 8.0 +/-0.1;
4) and uniformly treating the prepared treatment solution on glass fiber, and drying for 8-24 hours in a drying oven or drying room at 37 ℃.
Example 3 treponema pallidum antibody detection reagent (latex method) product sample pad treatment
The preparation steps of the sample pad are as follows:
1) taking purified water with the amount of 0.9 required by the treatment solution, sequentially adding 0.04-0.06M of Tris, 0.15-0.25 mM of Casein and 0.15-0.35 mM of PVP into the purified water, stirring the former component for complete dissolution, adding the next component for stirring for complete dissolution, and clarifying the solution;
2) adding the following components in the solution prepared in the step 1) in sequence: 0.01-0.015M of Na2CO30.2-0.6% of surfactant in the solution to be treated, stirring the former component to be completely dissolved, adding the next component, stirring to be completely dissolved, and clarifying the solution;
3) adjusting the pH value of the solution to 8.0 +/-0.1 by using hydrochloric acid or sodium hydroxide;
4) adding the following components into the solution with the adjusted pH value: 3-5% of mouse-derived anti-RBC in the required treatment solution is stirred until the solution is clear;
5) fixing the volume of the solution to be treated by using purified water, and repeatedly measuring the pH value to be 8.0 +/-0.1;
6) and uniformly treating the prepared treatment solution on glass fiber, and drying for 8-24 hours in a drying oven or drying room at 37 ℃.
Example 4 helicobacter pylori IgG antibody detection reagent (latex method) product blood filtration sample pad treatment
The preparation steps of the sample pad are as follows:
1) taking purified water with the amount of 0.8 required by the treatment solution, adding 0.08-0.12M of Tris, 0.3-0.5 mM of PVP, 0.02-0.04M of ethylene diamine tetraacetic acid, 8-12% of protein stabilizer, 0.2-0.6% of surfactant and 0.15-0.25 mM of Casein into the solution to be treated according to the following sequence, adding the next component after the former component is completely dissolved by stirring, stirring until the next component is completely dissolved, and clarifying the solution;
2) adjusting the pH value of the solution to 8.0 +/-0.1 by using hydrochloric acid or sodium hydroxide;
3) adding 3-5% of murine anti-RBC to the solution with the adjusted pH value, and stirring until the solution is clear;
4) fixing the volume of the solution to be treated, and repeatedly measuring the pH value to be 8.0 +/-0.1;
5) and uniformly treating the prepared treatment solution on glass fiber, and drying for 8-24 hours in a drying oven or drying room at 37 ℃.
In summary, the sample pad selects different biological sources for the anti-RBC used for different product performances. For example, the human immunodeficiency virus (HIV 1/2) antibody detection reagent (latex method) selects murine anti-RBC, and the hepatitis B virus surface antigen detection reagent (colloidal gold method) selects ovine anti-RBC.
The surfactant has a washing effect, can dissolve a marker, enhance the water solubility of protein, eliminate non-specific binding (covering some miscellaneous sites), and simultaneously has a certain carrier effect; PVP can eliminate non-specific adsorption and has a certain effect of a surfactant; protein substances such as Casein and the like can specifically adsorb hybrid protein (non-specific protein) and eliminate false positive or false negative in the detection process; adjusting the pH to 8.0. + -. 0.1 may eliminate false positives and may sometimes alter the protein conformation, allowing more binding sites to be exposed and thus more available for binding to the label, resulting in increased sensitivity.
The prepared treatment solution is uniformly treated on the glass fiber and is dried for 8-24 hours in a drying oven or drying room at 37 ℃, so that the solution can be better attached to the glass fiber, and the performance of the blood filtration sample pad is not influenced.
Preparation and detection results of the rapid diagnostic reagent strip:
the rapid diagnosis reagent strip comprises a blood filtration sample pad, a marking pad, an NC membrane and a water absorption pad, wherein the blood filtration sample pad is a sample adding position of a detected sample, can filter and buffer the detected sample, and reduces the interference of the ionic strength or the pH value in the sample on the detection; the recombinant antigen marker fixed by drying is arranged on the marking pad, and the antibody in the sample can be traced and reacts with the antibody to form the antibody-recombinant antigen marker, so that the antibody in the sample carries color; the NC membrane is coated with a detection line and a quality control line in advance, and can be combined with an antibody-recombinant antigen marker through immunoreaction, the combined marker is gathered at the detection line and the quality control line, and the result is interpreted according to the color development condition of the detection line; the water absorption pad can drive the antibody-recombinant antigen marker formed on the marker pad to move upwards through the upward flow of the liquid sample under the water absorption effect, so that the antibody-recombinant antigen marker reacts with the recombinant antigen detected out of the detection line.
The blood filtering sample pad, the marking pad, the NC membrane and the water absorption pad 4 are assembled in sequence, and the running board background is clean and has no abnormity when a product using the formula of the blood filtering sample pad keeps excellent test functionality (sensitivity, specificity and the like), so that the production cost is saved to a great extent, and the working efficiency is improved.
The rapid diagnostic reagent strip produced by using the blood filtration sample pad formula and the preparation process obtains the following results through a large number of experiments and evidence collection:
the data show that the background of the rapid diagnostic reagent strip after sample application by using the blood filtration sample pad is clean and has no phenomena of blood residue trace, no floating, no line whitening, no track and the like.
The above description is illustrative and not restrictive. Many modifications and variations of the present invention will be apparent to those skilled in the art in light of the above teachings, which will fall within the spirit and scope of the invention.
Claims (5)
1. A blood filtration sample pad comprises glass fibers and a treatment solution of the glass fibers, wherein the treatment solution comprises purified water, anti-RBC, a surfactant, a phosphate buffer solution, tris (hydroxymethyl) aminomethane, casein and polyvinylpyrrolidone, and is characterized by further comprising ethylene diamine tetraacetic acid, and the anti-RBC comprises murine anti-RBC or ovine anti-RBC.
2. The method of preparing a hemofilter sample pad according to claim 1, characterized in that the method of preparing the treatment solution is as follows:
1) taking purified water with the amount of 0.7 required by the treatment solution, sequentially adding 0.04-0.06M of tris (hydroxymethyl) aminomethane, 0.1-0.3 mM of polyvinylpyrrolidone, 0.02-0.1% of surfactant, 0.15-0.25 mM of casein, 0.02-0.04M of ethylenediamine tetraacetic acid, 8-12% of protein stabilizer of the required treatment solution and 2-5% of ovine anti-RBC of the required treatment solution into the purified water, stirring the former component to be completely dissolved, adding the next component, and stirring to be completely dissolved;
2) adjusting the pH value of the solution to 8.0 +/-0.1;
3) the solution with the adjusted pH value is added with purified water to the required volume, and the pH value is measured again to be 8.0 +/-0.1.
3. The method of preparing a hemofilter sample pad according to claim 1, characterized in that the method of preparing the treatment solution is as follows:
1) taking purified water with the amount of 0.8 of the treatment solution, sequentially adding 0.08-0.12M of trihydroxymethyl aminomethane, 0.3-0.5 mM of polyvinylpyrrolidone, 0.02-0.04M of ethylene diamine tetraacetic acid, 8-12% of protein stabilizer, 0.2-0.6% of surfactant and 0.15-0.25 mM of casein into the purified water, stirring the former component to be completely dissolved, adding the next component, and stirring to be completely dissolved;
2) adjusting the pH value of the solution to 8.0 +/-0.1;
3) adding the following components into the solution with the adjusted pH value: 3-5% of mouse-derived anti-RBC in the required treatment solution is stirred until the solution is clear;
4) the solution is metered to the volume of the required treatment solution, and the pH value is measured again to be 8.0 +/-0.1.
4. The method of preparing a blood-filtered sample pad according to claim 2 or 3, wherein the pH adjusting solution is either hydrochloric acid or sodium hydroxide solution.
5. The method of preparing a hemofilter sample pad according to claim 1 or 2 or 3 or 4, characterized by comprising the steps of:
preparing the treatment solution;
uniformly treating the prepared treatment solution on glass fibers;
and (3) drying the glass fiber treated by the treatment solution for 8-24 hours at 37 ℃.
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| CA2104976A1 (en) * | 1992-09-02 | 1994-03-03 | Stephan D. Daubney | Separation of plasma or serum from whole blood using a red blood cell binding component and a polymer containing multiple cationic sites |
| CN1110313A (en) * | 1994-04-15 | 1995-10-18 | 南京红十字血液中心 | Glass fibre leucocyte filter |
| JPH09196911A (en) * | 1996-01-19 | 1997-07-31 | Fuji Photo Film Co Ltd | Blood filter unit |
| CN101469323A (en) * | 2007-12-28 | 2009-07-01 | 英科新创(厦门)科技有限公司 | Method for separating red corpuscle from whole blood |
| CN101750244B (en) * | 2008-10-13 | 2014-03-12 | 艾博生物医药(杭州)有限公司 | Method for separating red cells from blood sample and application |
| CN101862564B (en) * | 2010-03-16 | 2012-03-21 | 苏州市玮琪生物科技有限公司 | Micro-blood sample pretreatment system and preparation process thereof |
| CN104111333A (en) * | 2013-11-13 | 2014-10-22 | 叶森 | Fast erythrocyte removal method applicable to whole blood detection |
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