CN109142759B - Preparation method of high-quality microcolumn gel for blood type detection card - Google Patents
Preparation method of high-quality microcolumn gel for blood type detection card Download PDFInfo
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- CN109142759B CN109142759B CN201811006993.8A CN201811006993A CN109142759B CN 109142759 B CN109142759 B CN 109142759B CN 201811006993 A CN201811006993 A CN 201811006993A CN 109142759 B CN109142759 B CN 109142759B
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- 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
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Abstract
The invention relates to a preparation method of a microcolumn gel for a high-quality blood type detection card, which comprises the steps of screening polyacrylamide sephadex, washing the polyacrylamide sephadex, soaking the polyacrylamide sephadex and washing the polyacrylamide sephadex again. The preparation method of the microcolumn gel for the high-quality blood type detection card can prepare and produce the microcolumn gel for the high-quality blood type detection card, and the blood type detection card produced by the microcolumn gel prepared by the method can obviously improve the sensitivity and specificity of the product.
Description
Technical Field
The invention relates to a preparation method of a microcolumn gel for a high-quality blood type detection card.
Background
The milestone event in the history of blood transfusion was the discovery of A, B, O blood types by Karl Landerstein et al, university of Vienna, Olympic 1900. Two years later, his student again discovered blood type AB, which opened a new era of safe blood transfusion, and Landersteiner therefore won the nobel prize in 1930. The MNS, P and Rh systems are discovered in succession in 1927-1947, and a safe blood transfusion technology is primarily established.
Clinically effective blood firstly requires a clinician to determine which patients need blood transfusion under which conditions, effectively treats blood coagulation disorder and prevents adverse reactions of blood transfusion, and clinical blood transfusion technical specifications issued by the ministry of health of China in 2000 require that ABO blood types of blood recipients and blood donors are reviewed before blood transfusion, and Rh blood types of patients are routinely checked, and the like.
The common methods for blood type detection include classical test tube method, microcolumn gel method, microplate method, slide method, etc., wherein the microcolumn gel method is an advanced and popular inspection technology at present, and is a novel blood type detection method combining technologies of erythrocyte membrane antigen and antibody combination, molecular sieve technology in gel medium, centrifugation technology, etc. Compared with other methods, the micro-column gel technology has the advantages of higher test sensitivity, more accurate result, standardized operation, good repeatability, stable result, easy mastering and reduced experiment error. At present, the micro-column gel products in the market are different, the core of the preparation of the high-quality blood type micro-column gel card is the preparation of the micro-column gel, and through retrieval, no paper and patent aiming at the core technology of the micro-column gel exist at present, and the micro-column gel in the market has poor specificity when screening free molecules, so that the sensitivity is not high.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a preparation method of a high-quality microcolumn gel for a blood type detection card, which is high in sensitivity and good in specificity.
The purpose of the invention is realized as follows:
a preparation method of a microcolumn gel for a high-quality blood type detection card comprises the following steps:
step one, screening polyacrylamide sephadex
Selecting polyacrylamide dextran gel with the grain diameter of 10-100 nanometers being more than or equal to 65%, and soaking the gel in 20ml of normal saline for 24-48 hours per 1 g of gel;
step two, washing of polyacrylamide sephadex
Fully and uniformly mixing the sephadex swelling glue, centrifuging for 2 minutes by using a centrifugal force of 900g, and sucking a supernatant; adding equal volume of 0.9% sodium chloride solution, and repeating the operation for 3-5 times until the upper layer has no fine particles;
step three, soaking the polyacrylamide sephadex
The formula of the gel soak solution is as follows:
sodium chloride 2.5X 10-3g/ml
PEG400 (2.2-2.6)×10-3g/ml
Succinimide propionate 4X 10-5g/ml
Dissolving the above reagents with purified water, adjusting pH to 7.2-7.4, and soaking the washed dextran gel for 15-30 min;
step four, rewashing the polyacrylamide sephadex
The formula of the gel washing solution is as follows:
dissolving the above reagents with purified water, adjusting pH to 6.6-6.8, and washing gel for 3-5 times at a volume ratio of 1:1 with the gel washing solution to obtain gel particles with uniform particles.
Detailed Description
Step one, screening polyacrylamide sephadex
The gel is selected to be polyacrylamide dextran gel, the proportion of the gel particle size of 10-100 nanometers is more than or equal to 65%, and each 1 g of gel is soaked in 20ml of normal saline for 24-48 hours to ensure that the gel is fully expanded.
Step two, washing of polyacrylamide sephadex
The fully swollen polyacrylamide sepharose is washed to remove broken gel pieces and fine particles from the solution. The specific method comprises the following steps: the Sephadex swollen gel was thoroughly mixed, centrifuged for 2 minutes using a centrifugal force of 900g, and the supernatant was aspirated. An equal volume of 0.9% sodium chloride solution was added and the procedure was repeated 3-5 times until the upper layer was free of fine particles.
Step three, soaking the polyacrylamide sephadex
The formula of the gel soak solution is as follows:
sodium chloride 2.5X 10-3g/ml
PEG400 (2.2-2.6)×10-3g/ml
Succinimide propionate 4X 10-5g/ml
The above reagents are dissolved in purified water and the pH is adjusted to 7.2-7.4. PEG400 has wide compatibility, and the succinimide propionate can modify the PEG400 and effectively increase the solubility of other substances. The gel soaking solution is used for soaking the washed glucan gel for 15-30 minutes, so that the gel can be further swelled, and the particle size is more uniform.
Step four, rewashing the polyacrylamide sephadex
The formula of the gel washing solution is as follows:
dissolving the above reagents with purified water, and adjusting pH to 6.6-6.8; the methyl p-hydroxybenzoate and propyl p-hydroxybenzoate have phenolic hydroxyl structures, which can destroy the cell membrane of microorganism, change the protein value in the cell, and effectively inhibit the growth of microorganism; the dipotassium ethylenediamine tetraacetate can form chelate with calcium ions in blood, thereby preventing non-specific coagulation of blood and improving the sensitivity and specificity of the product. Bovine serum albumin has immunogenicity, but the modified bovine serum albumin by the monomethoxy polyethylene glycol succinimide carbonate can reduce the immunocompetence, improve the agglutination effect and avoid the trailing phenomenon. The gel washing solution is used for washing gel for 3-5 times according to the volume ratio of 1:1, so that gel particles with uniform particles can be obtained, and the gel washing solution can be used for immunoassay of microcolumn gel.
Compared with the prior art, the invention has the advantages that: the GEG400 has wide compatibility, can effectively increase the solubility of other substances, slows down the speed of molecules passing through the gel, and increases the reaction time. The methyl p-hydroxybenzoate and propyl p-hydroxybenzoate have phenolic hydroxyl structures which can destroy the cell membrane of microorganism, change the protein value in cells, effectively inhibit the growth of microorganism, and prevent gel or antibody from losing efficacy due to bacterial reproduction; the dipotassium ethylene diamine tetraacetate can form chelate with calcium ions in blood, so that non-specific coagulation of the blood is prevented, and the sensitivity and specificity of the product are improved; PEG400 and bovine serum albumin are used as buffer liquid, the agglutination effect is good, and no tailing condition occurs. The modified PEG can not specifically modify protein, but overcomes the defect by adding the dipotassium ethylenediamine tetraacetate; bovine serum albumin has immunogenicity, but the modified PEG modifies bovine serum albumin again to reduce the immunocompetence thereof, improve the agglutination effect, and avoid the trailing phenomenon, so that the bovine serum albumin has specificity and sensitivity, the reaction time is prolonged, and the experimental effect is obviously increased.
Claims (1)
1. A preparation method of a microcolumn gel for a high-quality blood type detection card is characterized by comprising the following steps: the preparation method comprises the following steps:
step one, screening polyacrylamide sephadex
Selecting polyacrylamide dextran gel with the grain diameter of 10-100 nanometers being more than or equal to 65%, and soaking the gel in 20ml of normal saline for 24-48 hours per 1 g of gel;
step two, washing of polyacrylamide sephadex
Fully and uniformly mixing the sephadex swelling glue, centrifuging for 2 minutes by using a centrifugal force of 900g, and sucking a supernatant; adding equal volume of 0.9% sodium chloride solution, and repeating the operation for 3-5 times until the upper layer has no fine particles;
step three, soaking the polyacrylamide sephadex
The formula of the gel soak solution is as follows:
sodium chloride 2.5X 10-3g/ml
PEG400 (2.2-2.6)×10-3g/ml
Succinimide propionate 4X 10-5g/ml
Dissolving the above reagents with purified water, adjusting pH to 7.2-7.4, and soaking the washed dextran gel for 15-30 min;
step four, rewashing the polyacrylamide sephadex
The formula of the gel washing solution is as follows:
potassium dihydrogen phosphate (2.3-2.5) x 10-4 g/ml
Disodium hydrogen phosphate (4.4-5.0). times.10-4 g/ml
Sodium chloride (1.7-2.0). times.10-3g/ml
Glycine (1.6-1.8) x 10-2g/ml
Methylparaben (5.5-6.5). times.10-4 g/ml
Propyl p-hydroxybenzoate (1.2-1.6). times.10-4 g/ml
Ethylene diamine tetraacetic acid dipotassium (5.0-5.5) multiplied by 10-3g/ml
Bovine serum albumin (0.8-1.2). times.10-3g/ml
Dissolving the above reagents with purified water, adjusting pH to 6.6-6.8, and washing gel for 3-5 times at a volume ratio of 1:1 with the gel washing solution to obtain gel particles with uniform particles.
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