CN112285340A - Latex immunoturbidimetry method of alpha 1-acid glycoprotein detection kit and preparation method thereof - Google Patents
Latex immunoturbidimetry method of alpha 1-acid glycoprotein detection kit and preparation method thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biology, in particular to an alpha 1-acid glycoprotein detection kit latex immunoturbidimetry, and further relates to a preparation method of the alpha 1-acid glycoprotein detection kit latex immunoturbidimetry, and the alpha 1-acid glycoprotein detection kit latex immunoturbidimetry is characterized in that: the ratio of the biotin to the alpha 1-acid glycoprotein monoclonal antibody is 1:1-3:1, and the ratio of the streptavidin to the biotin-alpha 1-acid glycoprotein monoclonal antibody is 0.5:1-4: 1. The advantages are that: after a plurality of research and development experiments, the streptavidin 1:1 is connected by latex microspheres, the alpha 1-acid glycoprotein antibody is connected with the biotin 1:2, and the two are mixed for reaction at the ratio of 1:1, so that the repeatability and the sensitivity can be obviously improved, and the linear range can be 0.00-3.00 g/L.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an alpha 1-acid glycoprotein detection kit latex immunoturbidimetry and a preparation method of the alpha 1-acid glycoprotein detection kit latex immunoturbidimetry.
Background
Alpha 1-acid glycoprotein is a nonspecific acute phase reaction protein, and is glycoprotein with the highest sugar content and the strongest acidity in human plasma. The alpha 1-acid glycoprotein is composed of polypeptide chains consisting of 181 amino acid residues, has a single-chain molecular structure, contains about 40 percent of sugar, comprises equal molecules of hexose, hexosamine and sialic acid, and is positioned at the alpha 1 position during electrophoretic movement, so the alpha 1-acid glycoprotein is called, has the molecular weight of about 40000KD and the pH value of 2.7-3.5.
Alpha 1-acid glycoprotein is produced mainly by liver macrophages and granulocytes, and is also synthesized by some cancer cells. Early alpha 1-acid glycoprotein is widely recognized as a non-specific tumor marker, and a plurality of studies later show that the concentration of the alpha 1-acid glycoprotein in patients with nephropathy, gastropathy and the like is increased, so that the research index is reduced. The content of alpha 1-acid glycoprotein in normal human serum is low, and the concentration of the alpha 1-acid glycoprotein is obviously increased under the pathological conditions of infection, inflammation, tumor and the like. In addition, α 1-acid glycoprotein is also considered as a major component of mucin, but currently, due to the complicated operation of mucin, many factors, and low sensitivity of detection results, it has been proposed to be eliminated. In recent years, with the continuous improvement of the detection technology and the detection equipment, the detection sensitivity of the alpha 1-acid glycoprotein is remarkably improved, the detection method and the standardization of the measurement result are gradually implemented, the clinical application starts to have new knowledge on the alpha 1-acid glycoprotein, and the detection on the alpha 1-acid glycoprotein is increasingly emphasized. Foreign academic journals report that the concentration of alpha 1-acid glycoprotein in serum is obviously increased when breast cancer, ovarian cancer and other pathological changes occur.
In recent years, the clinical value of α 1-acid glycoprotein has been receiving much attention. Research shows that the concentration of alpha 1-acid glycoprotein in serum of malignant tumor patients is obviously increased and is related to the treatment process of the patients. In addition, researches show that the determination of the ascites alpha 1-acid glycoprotein not only can identify exudates and effusions, but also is beneficial to the identification of benign and malignant ascites. The previous study on the concentration of alpha 1-acid glycoprotein in healthy people and the correlation between the concentration and the age indicates that the higher serum concentration is rare despite the wider range of the concentration of alpha 1-acid glycoprotein listed in the relevant literature, and the fact that the higher serum concentration of alpha 1-acid glycoprotein in the serum is possibly predictive of the existence of a certain disease or pathological state as a non-specific laboratory index is suggested. Since hepatic synthesis and release of α 1-acid glycoprotein are regulated by various hormones in vivo, especially sex hormones, estrogen can inhibit the production of α 1-acid glycoprotein.
At present, the detection methods for alpha 1-acid glycoprotein mainly comprise radioimmunoassay and enzyme-linked immunosorbent assay. These detection methods have many disadvantages, such as the need for specific equipment, the need for sample pretreatment, and the inability to perform batch detection with fully automated biochemical analyzers. In addition, radioimmunoassay has problems with radiation and contamination. Enzyme-linked immunosorbent assay is widely applied, but the method is heterogeneous immunoassay, the determination process is complicated, the time consumption is long, the batch difference and the repeatability are relatively large, multiple special equipment needs to be equipped, and the cost is increased to a certain extent.
In view of the above, the invention provides a kit for detecting alpha 1-acid glycoprotein by an immunotransmission turbidimetric assay, in which latex microspheres are directly coupled with an alpha 1-acid glycoprotein antibody in a conventional method, and sensitivity and a linear range are relatively poor by using the method.
Disclosure of Invention
The purpose of the invention is: aiming at the defects, the latex immunoturbidimetry of the alpha 1-acid glycoprotein detection kit and the preparation method thereof are provided, wherein the repeatability and the sensitivity are improved.
In order to achieve the purpose, the invention adopts the technical scheme that:
the latex immunoturbidimetry of the alpha 1-acid glycoprotein detection kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following main components in concentration:
the main components of the reagent R2 comprise: a second buffer solution, a preservation solution and a latex microsphere antibody conjugate of the reagent R2, wherein the preservation solution comprises N, N-dihydroxyethyl glycine, a stabilizing agent and a second preservative; the latex microsphere antibody conjugate comprises latex microspheres, biotin, streptavidin, an alpha 1-acid glycoprotein monoclonal antibody and 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride; the concentration of the main components is as follows:
the further technical scheme is as follows:
the first buffer solution A and the second buffer solution B of the reagent R1 are PBS buffer solutions.
The promoter of the reagent R1 is PEG 6000.
The preservative of the reagent R1 is one or more of sodium azide, Proclin-950 and Proclin-300.
The second buffer in the reagent R2 was MES buffer.
The stabilizer in the reagent R2 is bovine serum albumin.
The second preservative in the reagent R2 is one of sodium azide, Proclin-950 and Proclin-300.
The ratio of the biotin to the alpha 1-acid glycoprotein monoclonal antibody is 1:1-3:1, and the ratio of the streptavidin to the biotin-alpha 1-acid glycoprotein monoclonal antibody is 0.5:1-4: 1.
A preparation method of an alpha 1-acid glycoprotein detection kit by a latex immunoturbidimetry method comprises the following steps: A) reagent R1 preparation:
adding part of purified water into a liquid preparation tank, adding a first buffer solution A and a second buffer solution A while stirring until the materials are completely dissolved, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adding sodium chloride, sequentially adding an accelerant and a first preservative according to the feeding mode after the materials are completely dissolved, continuously stirring until all the materials are completely dissolved, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value to be 6.80-8.00, and fixing the volume to the final volume;
filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of a microporous filter, storing the filtered solution in a finished product tank, and marking;
B) reagent R2 preparation:
step B1: adding part of purified water into the liquid preparation tank on a magnetic stirrer, adjusting the rotating speed of the stirrer to 200-300rpm to keep the stirring in a uniform speed state, adding a second buffer solution while stirring until the materials are completely dissolved, keeping the materials clear and transparent, adjusting the pH value to 4.50-6.50 without sediment at the bottom of the liquid preparation tank, and fixing the volume to the final volume;
filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of a microporous filter, storing the filtered solution in a finished product tank, and marking;
step B2: preparation of preserving fluid
Adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting the rotating speed of the stirrer to 200-300rpm to keep stirring in a uniform speed state, adding N, N-dihydroxyethyl glycine while stirring until the material is completely dissolved, adding a stabilizer, adding a second preservative after the material is completely dissolved, keeping the solution clear and transparent, adjusting the pH value to 6.30-8.50 without sediment at the bottom of the liquid preparation tank, and fixing the volume to the final volume;
filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of the microporous filter, and storing the filtered solution for later use for marking;
step B3: preparation of latex microsphere antibody conjugates
Diluting latex microspheres to the concentration of 0.3-3.0% by using a reaction solution, combining the latex microspheres with streptavidin according to a certain proportion, diluting an alpha 1-acid glycoprotein monoclonal antibody to the concentration of 0.05-3.0g/L by using the reaction solution, dialyzing the mixture with biotin according to a certain proportion overnight, adding the mixture into microspheres containing the streptavidin, uniformly mixing, dissolving 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride into the concentration of 0.005-0.3g/L by using the reaction solution, shaking uniformly, dropwise adding the mixture into the mixed solution while reacting for 120 minutes at room temperature, adding a stabilizing agent for sealing, oscillating and uniformly mixing, reacting for 60 minutes at room temperature, centrifuging the mixed solution for 30 minutes at the rotating speed of 14000rpm, sucking off a supernatant, adding a preserving solution, carrying out ultrasonic resuspension, repeatedly centrifuging and cleaning for 3 times, finally centrifuging once to remove the supernatant, adding preservation solution, carrying out ultrasonic resuspension, filling the prepared R2 into a finished product tank, and fixing the volume to the final concentration for marking.
Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages: after a plurality of research and development experiments, the streptavidin 1:1 is connected by latex microspheres, the alpha 1-acid glycoprotein antibody is connected with the biotin 1:2, and the two are mixed for reaction at the ratio of 1:1, so that the repeatability and the sensitivity can be obviously improved, and the linear range can be 0.00-3.00 g/L.
Drawings
FIG. 1 is a calibration curve of the direct coupling of latex microspheres to alpha 1-acid glycoprotein antibodies of example 5 of the present invention. Where the X-axis represents standard concentration and the Y-axis represents dOD.
FIG. 2 is a graph showing the calibration curve of the indirect coupling of latex microspheres to alpha 1-acid glycoprotein antibodies of example 6 of the present invention. Where the X-axis represents standard concentration and the Y-axis represents dOD.
Detailed Description
The invention is further described with reference to the following figures and examples:
the first embodiment is as follows:
the latex immunoturbidimetry of the alpha 1-acid glycoprotein detection kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following main components in concentration: the concentration of the sodium dihydrogen phosphate dihydrate serving as a first buffer solution A is 0.8g/L, the concentration of the disodium hydrogen phosphate dodecahydrate serving as a second buffer solution A is 8.75g/L, the concentration of sodium chloride is 5.8g/L, the concentration of PEG 6000 serving as an accelerant is 40g/L, and the concentration of Proclin-950 serving as a first preservative is 0.8 ml/L;
the reagent R2 comprises the following main components in percentage by weight: a second buffer solution, a preservation solution and a latex microsphere antibody conjugate of the reagent R2, wherein the concentration of 2- (N-morpholine) ethanesulfonic acid monohydrate used as the second buffer solution is 8.05g/L, the preservation solution comprises N, N-dihydroxyethylglycine, the concentration of N, N-dihydroxyethylglycine is 1.26g/L, bovine serum albumin used as a stabilizing agent is 0.5g/L, and Proclin-950 used as a second preservative is 0.8 ml/L; the latex microsphere antibody conjugate comprises latex microspheres, the concentration of which is 0.05 percent, biotin, the concentration of which is 5g/L, streptavidin, the concentration of which is 5g/L, alpha 1-acid glycoprotein monoclonal antibody, the concentration of which is 0.05g/L and 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride, and the concentration of which is 0.005 g/L.
The ratio of the biotin to the alpha 1-acid glycoprotein monoclonal antibody is 2:1, and the ratio of the streptavidin to the biotin-alpha 1-acid glycoprotein monoclonal antibody is 1: 1.
A preparation method of an alpha 1-acid glycoprotein detection kit by a latex immunoturbidimetry method comprises the following steps: A) reagent R1 preparation:
adding part of purified water into a liquid preparation tank, adding part of purified water into a magnetic stirrer, adjusting the rotating speed of the stirrer to 200rpm, keeping the stirring in a constant speed state, adding sodium dihydrogen phosphate dihydrate and disodium hydrogen phosphate dodecahydrate while stirring until the materials are completely dissolved, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adding sodium chloride, sequentially adding PEG 6000 and Proclin-950 according to the feeding mode after the materials are completely dissolved, continuously stirring until all the materials are completely dissolved after the feeding is finished, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value to 6.80, and fixing the volume to the final volume;
filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of a microporous filter, storing the filtered solution in a finished product tank, and marking;
B) reagent R2 preparation:
step B1: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting the rotating speed of the stirrer to 200rpm, keeping the stirring in a constant speed state, adding 2- (N-morpholine) ethanesulfonic acid monohydrate while stirring, stirring until the materials are completely dissolved, keeping the materials clear and transparent, ensuring that no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value to 4.50, and fixing the volume to the final volume;
filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of a microporous filter, storing the filtered solution in a finished product tank, and marking;
step B2: preparation of preserving fluid
Adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting the rotating speed of the stirrer to 200rpm, keeping the stirring in a constant speed state, adding N, N-dihydroxyethylglycine while stirring until the material is completely dissolved, adding a stabilizer, adding bovine serum albumin after the material is completely dissolved, keeping the volume to be the final volume after the bovine serum albumin is clear and transparent and no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value to 6.30 and fixing the volume to the final volume;
filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of the microporous filter, and storing the filtered solution for later use for marking;
step B3: preparation of latex microsphere antibody conjugates
Diluting latex microspheres to 0.3% concentration by using reaction solution, combining the latex microspheres with streptavidin according to a ratio of 1:1, diluting alpha 1-acid glycoprotein monoclonal antibody to 0.05g/L concentration by using reaction solution, combining the reaction solution with biotin according to a ratio of 1:2, dialyzing overnight, and then performing dialysis according to a ratio of 1:1, adding the mixture into microspheres containing streptavidin, uniformly mixing, dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into a reaction solution to a concentration of 0.005g/L, dropwise adding the reaction solution into the mixed solution while shaking uniformly, reacting for 120 minutes at room temperature, adding a stabilizer for sealing, oscillating and uniformly mixing, reacting for 60 minutes at room temperature, centrifuging the mixed solution for 30 minutes at a rotating speed of 14000rpm, sucking off supernatant, adding preservation solution, ultrasonically resuspending, repeatedly centrifuging and cleaning for 3 times, centrifuging the supernatant for the last time, adding the preservation solution, ultrasonically resuspending, filling the prepared R2 into a finished product tank, fixing the volume to the final concentration, and marking.
Example two:
the latex immunoturbidimetry of the alpha 1-acid glycoprotein detection kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following main components in concentration: the concentration of the sodium dihydrogen phosphate dihydrate serving as a first buffer solution A is 1.6g/L, the concentration of the disodium hydrogen phosphate dodecahydrate serving as a second buffer solution A is 14g/L, the concentration of the sodium chloride is 10g/L, the concentration of the PEG 6000 serving as an accelerating agent is 70g/L, and the concentration of the Proclin-300 serving as a first preservative is 1.2 ml/L;
the reagent R2 comprises the following main components in percentage by weight: a second buffer solution, a preservation solution and a latex microsphere antibody conjugate of the reagent R2, wherein the concentration of 2- (N-morpholine) ethanesulfonic acid monohydrate serving as the second buffer solution is 14g/L, the preservation solution comprises N, N-dihydroxyethylglycine, the concentration of 4g/L, bovine serum albumin serving as a stabilizing agent, the concentration of 1g/L and Proclin-300 serving as a second preservative, and the concentration of 1.2 ml/L; the latex microsphere antibody conjugate comprises latex microspheres, the concentration of which is 0.6 percent, biotin, the concentration of which is 10g/L, streptavidin, the concentration of which is 10g/L, alpha 1-acid glycoprotein monoclonal antibody, the concentration of which is 1g/L and 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride, and the concentration of which is 0.1 g/L.
The ratio of the biotin to the alpha 1-acid glycoprotein monoclonal antibody is 2:1, and the ratio of the streptavidin to the biotin-alpha 1-acid glycoprotein monoclonal antibody is 1: 1.
A preparation method of an alpha 1-acid glycoprotein detection kit by a latex immunoturbidimetry method comprises the following steps: A) reagent R1 preparation:
adding part of purified water into a liquid preparation tank, adding part of purified water into a magnetic stirrer, adjusting the rotating speed of the stirrer to 225rpm, keeping the stirring in a constant speed state, adding sodium dihydrogen phosphate dihydrate and disodium hydrogen phosphate dodecahydrate while stirring until the materials are completely dissolved, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adding sodium chloride, sequentially adding PEG 6000 and Proclin-300 according to the feeding mode after the materials are completely dissolved, continuously stirring until all the materials are completely dissolved after the feeding is finished, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value to 7.2, and fixing the volume to the final volume;
filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of a microporous filter, storing the filtered solution in a finished product tank, and marking;
B) reagent R2 preparation:
step B1: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting the rotating speed of the stirrer to 225rpm, keeping the stirring in a constant speed state, adding 2- (N-morpholine) ethanesulfonic acid monohydrate while stirring, stirring until the materials are completely dissolved, keeping the materials clear and transparent, ensuring that no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value to 5, and fixing the volume to the final volume;
filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of a microporous filter, storing the filtered solution in a finished product tank, and marking;
step B2: preparation of preserving fluid
Adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting the rotating speed of the stirrer to 225rpm, keeping the stirring in a constant speed state, adding N, N-dihydroxyethylglycine while stirring until the material is completely dissolved, then adding bovine serum albumin, adding Proclin-300 after the material is completely dissolved, and adjusting the pH value to 7 and fixing the volume to the final volume when the material is clear and transparent and the bottom of the liquid preparation tank has no precipitate;
filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of the microporous filter, and storing the filtered solution for later use for marking;
step B3: preparation of latex microsphere antibody conjugates
Diluting the latex microspheres to the concentration of 1% by using a reaction solution, combining the latex microspheres with streptavidin according to the ratio of 1:1, diluting the alpha 1-acid glycoprotein monoclonal antibody to the concentration of 1g/L by using the reaction solution, combining the reaction solution with biotin according to the ratio of 1:2, dialyzing overnight, and then, mixing the diluted latex microspheres with the biotin according to the ratio of 1:1, adding the mixture into microspheres containing streptavidin, uniformly mixing, dissolving 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into a reaction solution to a concentration of 0.1g/L, dropwise adding the reaction solution into the mixed solution while shaking uniformly, reacting for 120 minutes at room temperature, adding a stabilizer for sealing, oscillating and uniformly mixing, reacting for 60 minutes at room temperature, centrifuging the mixed solution for 30 minutes at a rotating speed of 14000rpm, sucking off supernatant, adding preservation solution, ultrasonically resuspending, repeatedly centrifuging and cleaning for 3 times, centrifuging the supernatant for the last time, adding the preservation solution, ultrasonically resuspending, filling the prepared R2 into a finished product tank, fixing the volume to the final concentration, and marking.
Example three:
the latex immunoturbidimetry of the alpha 1-acid glycoprotein detection kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following main components in concentration: the concentration of the sodium dihydrogen phosphate dihydrate serving as a first buffer solution A is 2.4g/L, the concentration of the disodium hydrogen phosphate dodecahydrate serving as a second buffer solution A is 18g/L, the concentration of the sodium chloride is 15g/L, the concentration of the PEG 6000 serving as an accelerating agent is 100g/L, and the concentration of the sodium azide serving as a first preservative is 1.6 ml/L;
the reagent R2 comprises the following main components in percentage by weight: a second buffer solution, a preservation solution and a latex microsphere antibody conjugate of the reagent R2, wherein the concentration of 2- (N-morpholine) ethanesulfonic acid monohydrate serving as the second buffer solution is 20g/L, the preservation solution comprises N, N-dihydroxyethylglycine, bovine serum albumin serving as a stabilizer, the concentration of 1.5g/L and sodium azide serving as a second preservative, and the concentration of the sodium azide is 1.6 ml/L; the latex microsphere antibody conjugate comprises latex microspheres, the concentration of which is 1.2 percent, biotin, the concentration of which is 15g/L, streptavidin, the concentration of which is 15g/L, alpha 1-acid glycoprotein monoclonal antibody, the concentration of which is 2g/L and 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride, and the concentration of which is 0.2 g/L.
The ratio of the biotin to the alpha 1-acid glycoprotein monoclonal antibody is 2:1, and the ratio of the streptavidin to the biotin-alpha 1-acid glycoprotein monoclonal antibody is 1: 1.
A preparation method of an alpha 1-acid glycoprotein detection kit by a latex immunoturbidimetry method comprises the following steps: A) reagent R1 preparation:
adding part of purified water into a liquid preparation tank, adding part of purified water into a magnetic stirrer, adjusting the rotating speed of the stirrer to 260rpm, keeping the stirring in a constant speed state, adding sodium dihydrogen phosphate dihydrate and disodium hydrogen phosphate dodecahydrate while stirring until the materials are completely dissolved, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adding sodium chloride, sequentially adding PEG 6000 and sodium azide according to the feeding mode after the materials are completely dissolved, continuously stirring until all the materials are completely dissolved after the feeding is finished, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value to 7.2, and fixing the volume to the final volume;
filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of a microporous filter, storing the filtered solution in a finished product tank, and marking;
B) reagent R2 preparation:
step B1: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting the rotating speed of the stirrer to 260rpm, keeping the stirring in a constant speed state, adding 2- (N-morpholine) ethanesulfonic acid monohydrate while stirring, stirring until the materials are completely dissolved, keeping the materials clear and transparent, ensuring that no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value to 6, and fixing the volume to the final volume;
filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of a microporous filter, storing the filtered solution in a finished product tank, and marking;
step B2: preparation of preserving fluid
Adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting the rotating speed of the stirrer to 260rpm, keeping the stirring in a constant speed state, adding N, N-dihydroxyethyl glycine while stirring until the material is completely dissolved, then adding bovine serum albumin, adding sodium azide after the material is completely dissolved, keeping the solution clear and transparent, adjusting the pH value to 8 without sediment at the bottom of the liquid preparation tank, and fixing the volume to the final volume;
filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of the microporous filter, and storing the filtered solution for later use for marking;
step B3: preparation of latex microsphere antibody conjugates
Diluting latex microspheres to the concentration of 2% by using a reaction solution, combining the latex microspheres with streptavidin according to the proportion of 1:1, diluting an alpha 1-acid glycoprotein monoclonal antibody to the concentration of 2g/L by using the reaction solution, combining the reaction solution with biotin according to the proportion of 1:2, dialyzing overnight, adding the mixture into microspheres containing the streptavidin according to the proportion of 1:1, uniformly mixing, dissolving 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride into the concentration of 0.2g/L by using the reaction solution, uniformly shaking and dropwise adding the mixture into the mixed solution, reacting at room temperature for 120 minutes, adding a stabilizer for sealing, uniformly shaking, reacting at room temperature for 60 minutes, centrifuging the mixed solution at the rotating speed of 14000rpm for 30 minutes, sucking off a supernatant, adding a preservation solution, ultrasonically resuspending, repeatedly centrifuging and washing for 3 times, finally centrifuging the supernatant, adding the solution, and (3) carrying out ultrasonic resuspension, filling the prepared R2 into a finished product tank, and fixing the volume to the final concentration for marking.
Example four:
the latex immunoturbidimetry of the alpha 1-acid glycoprotein detection kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises the following main components in concentration: the concentration of the sodium dihydrogen phosphate dihydrate serving as a first buffer solution A is 3.2g/L, the concentration of the disodium hydrogen phosphate dodecahydrate serving as a second buffer solution A is 24.31g/L, the concentration of the sodium chloride is 20.0g/L, the concentration of the PEG 6000 serving as an accelerant is 120g/L, and the concentration of the Proclin-950 serving as a first preservative is 2.0 ml/L;
the reagent R2 comprises the following main components in percentage by weight: a second buffer solution, a preservation solution and a latex microsphere antibody conjugate of the reagent R2, wherein the concentration of 2- (N-morpholine) ethanesulfonic acid monohydrate used as the second buffer solution is 27.30g/L, the preservation solution comprises N, N-dihydroxyethylglycine, the concentration of 9.83g/L, bovine serum albumin used as a stabilizing agent, the concentration of 2.0g/L and Proclin-950 used as a second preservative, and the concentration of 2.0 ml/L; the latex microsphere antibody conjugate comprises latex microspheres, the concentration of which is 2.0 percent, biotin, the concentration of which is 20g/L, streptavidin, the concentration of which is 20g/L, alpha 1-acid glycoprotein monoclonal antibody, the concentration of which is 3g/L and 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride, and the concentration of which is 0.3 g/L.
The ratio of the biotin to the alpha 1-acid glycoprotein monoclonal antibody is 2:1, and the ratio of the streptavidin to the biotin-alpha 1-acid glycoprotein monoclonal antibody is 1: 1.
A preparation method of an alpha 1-acid glycoprotein detection kit by a latex immunoturbidimetry method comprises the following steps: A) reagent R1 preparation:
adding part of purified water into a liquid preparation tank, adding part of purified water into a magnetic stirrer, adjusting the rotating speed of the stirrer to 300rpm, keeping the stirring in a constant speed state, adding sodium dihydrogen phosphate dihydrate and disodium hydrogen phosphate dodecahydrate while stirring until the materials are completely dissolved, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adding sodium chloride, sequentially adding PEG 6000 and Proclin-950 according to the feeding mode after the materials are completely dissolved, continuously stirring until all the materials are completely dissolved after the feeding is finished, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value to 8.00, and fixing the volume to the final volume;
filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of a microporous filter, storing the filtered solution in a finished product tank, and marking;
B) reagent R2 preparation:
step B1: adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting the rotating speed of the stirrer to 300rpm, keeping the stirring in a constant speed state, adding 2- (N-morpholine) ethanesulfonic acid monohydrate while stirring, stirring until the materials are completely dissolved, keeping the materials clear and transparent, ensuring that no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value to 6.50, and fixing the volume to the final volume;
filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of a microporous filter, storing the filtered solution in a finished product tank, and marking;
step B2: preparation of preserving fluid
Adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting the rotating speed of the stirrer to 300rpm, keeping the stirring in a constant speed state, adding N, N-dihydroxyethylglycine while stirring until the material is completely dissolved, then adding bovine serum albumin, adding Proclin-950 after the material is completely dissolved, and adjusting the pH value to 8.50 and fixing the volume to the final volume when the material is clear and transparent and no precipitate is at the bottom of the liquid preparation tank;
filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of the microporous filter, and storing the filtered solution for later use for marking;
step B3: preparation of latex microsphere antibody conjugates
Diluting latex microspheres to the concentration of 3.0% by using a reaction solution, combining the latex microspheres with streptavidin according to the ratio of 1:1, diluting an alpha 1-acid glycoprotein monoclonal antibody to the concentration of 3.0g/L by using the reaction solution, combining the reaction solution with biotin according to the ratio of 1:2, dialyzing overnight, adding the mixture into microspheres containing the streptavidin according to the ratio of 1:1, uniformly mixing, dissolving 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride into the concentration of 0.3g/L by using the reaction solution, shaking uniformly, dropwise adding the mixture into the mixed solution, reacting at room temperature for 120 minutes, adding a stabilizer, sealing, shaking uniformly mixing, reacting at room temperature for 60 minutes, centrifuging the mixed solution at the rotating speed of 14000rpm for 30 minutes, removing a supernatant, adding a preservation solution, carrying out ultrasonic resuspension, repeatedly centrifuging and cleaning for 3 times, adding the preservation solution after the supernatant is removed by the last centrifugation, and (3) carrying out ultrasonic resuspension, filling the prepared R2 into a finished product tank, and fixing the volume to the final concentration for marking.
Example five:
the latex microspheres were directly coupled to the alpha 1-acid glycoprotein antibody and calibrated using standards, the results are shown in table 1, and the calibration curve is shown in fig. 1.
Table 1: the calibration result of the latex microsphere directly coupled with the alpha 1-acid glycoprotein antibody is as follows:
and (4) result analysis: as shown in FIG. 1 and Table 1, the linear range can be made 0.00-3.00g/L, and the hook effect appears at concentrations greater than 3.00 g/L.
The latex microspheres were indirectly coupled to the alpha 1-acid glycoprotein antibody and calibrated using standards, the results are shown in table 2, and the calibration curve is shown in fig. 2.
Table 2: the calibration result of the latex microsphere indirect coupling alpha 1-acid glycoprotein antibody is as follows:
and (4) result analysis: as shown in FIG. 2 and Table 2, the linear range can be made 0.00-5.00 g/L.
Example six:
and (3) precision CV detection:
the latex microspheres are directly coupled with the alpha 1-acid glycoprotein antibody, a sample is randomly taken, the same sample is repeatedly detected for 10 times, and the detection result is shown in table 3.
Table 3: the precision CV of the latex microsphere directly coupled with the alpha 1-acid glycoprotein antibody of the detection sample is as follows:
| 1 | 0.15 | 1.87 | 2.58 |
| 2 | 0.16 | 2.15 | 2.36 |
| 3 | 0.15 | 2.09 | 2.70 |
| 4 | 0.17 | 1.99 | 2.85 |
| 5 | 0.17 | 1.96 | 2.71 |
| 6 | 0.15 | 2.00 | 2.65 |
| 7 | 0.14 | 2.09 | 2.46 |
| 8 | 0.14 | 2.04 | 2.87 |
| 9 | 0.13 | 1.97 | 2.84 |
| 10 | 0.13 | 2.01 | 2.83 |
| mean value | 0.15 | 2.02 | 2.69 |
| SD | 0.01 | 0.08 | 0.17 |
| CV | 9.37％ | 3.90％ | 6.50％ |
And (4) result analysis: the precision CV is large and is basically more than 5 percent.
The latex microspheres are indirectly coupled with the alpha 1-acid glycoprotein antibody, a sample is randomly taken, the same sample is repeatedly detected for 10 times, and the detection result is shown in table 3.
Table 4: precision CV (constant-volume dilution) for indirectly coupling alpha 1-acid glycoprotein antibody by latex microspheres of detected sample
| 1 | 0.15 | 1.87 | 2.60 |
| 2 | 0.14 | 1.91 | 2.65 |
| 3 | 0.14 | 1.96 | 2.53 |
| 4 | 0.14 | 1.92 | 2.67 |
| 5 | 0.14 | 1.79 | 2.54 |
| 6 | 0.14 | 1.78 | 2.51 |
| 7 | 0.16 | 1.84 | 2.61 |
| 8 | 0.15 | 1.88 | 2.69 |
| 9 | 0.15 | 1.95 | 2.52 |
| 10 | 0.14 | 1.88 | 2.57 |
| Mean value | 0.15 | 1.88 | 2.59 |
| SD | 0.00 | 0.06 | 0.06 |
| CV | 3.30％ | 3.25％ | 2.50％ |
And (4) result analysis: the precision CV is obviously improved to be within 4 percent.
And (4) analyzing results: after a plurality of research and development experiments, the streptavidin 1:1 is connected by latex microspheres, the alpha 1-acid glycoprotein antibody is connected with the biotin 1:2, and the two are mixed for reaction at the ratio of 1:1, so that the repeatability and the sensitivity can be obviously improved, and the linear range can be 0.00-3.00 g/L.
The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Claims (9)
1. An alpha 1-acid glycoprotein detection kit latex immunoturbidimetry, which is characterized in that: the reagent R1 comprises a reagent R1 and a reagent R2, and the main components and the constituent concentration of the reagent R1 comprise:
the main components of the reagent R2 comprise: a second buffer solution, a preservation solution and a latex microsphere antibody conjugate of the reagent R2, wherein the preservation solution comprises N, N-dihydroxyethyl glycine, a stabilizing agent and a second preservative; the latex microsphere antibody conjugate comprises latex microspheres, biotin, streptavidin, an alpha 1-acid glycoprotein monoclonal antibody and 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride; the concentration of the main components is as follows:
2. the latex immunoturbidimetry of the alpha 1-acid glycoprotein assay kit of claim 1, wherein: the first buffer solution A and the second buffer solution B of the reagent R1 are PBS buffer solutions.
3. The latex immunoturbidimetry of the alpha 1-acid glycoprotein assay kit of claim 1, wherein: the promoter of the reagent R1 is PEG 6000.
4. The latex immunoturbidimetry of the alpha 1-acid glycoprotein assay kit of claim 1, wherein:
the preservative of the reagent R1 is one or more of sodium azide, Proclin-950 and Proclin-300.
5. The latex immunoturbidimetry of the alpha 1-acid glycoprotein assay kit of claim 1, wherein: the second buffer in the reagent R2 was MES buffer.
6. The latex immunoturbidimetry of the alpha 1-acid glycoprotein assay kit of claim 1, wherein: the stabilizer in the reagent R2 is bovine serum albumin.
7. The latex immunoturbidimetry of the alpha 1-acid glycoprotein assay kit of claim 1, wherein: the second preservative in the reagent R2 is one of sodium azide, Proclin-950 and Proclin-300.
8. The latex immunoturbidimetry of the alpha 1-acid glycoprotein assay kit of claim 1, wherein: the ratio of the biotin to the alpha 1-acid glycoprotein monoclonal antibody is 1:1-3:1, and the ratio of the streptavidin to the biotin-alpha 1-acid glycoprotein monoclonal antibody is 0.5:1-4: 1.
9. A preparation method of an alpha 1-acid glycoprotein detection kit by a latex immunoturbidimetry method is characterized by comprising the following steps: the method comprises the following steps: A) reagent R1 preparation:
adding part of purified water into a liquid preparation tank, adding a first buffer solution A and a second buffer solution A while stirring until the materials are completely dissolved, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adding sodium chloride, sequentially adding an accelerant and a first preservative according to the feeding mode after the materials are completely dissolved, continuously stirring until all the materials are completely dissolved, the solution is clear and transparent, no precipitate is left at the bottom of the liquid preparation tank, adjusting the pH value to be 6.80-8.00, and fixing the volume to the final volume;
filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of a microporous filter, storing the filtered solution in a finished product tank, and marking;
B) reagent R2 preparation:
step B1: adding part of purified water into the liquid preparation tank on a magnetic stirrer, adjusting the rotating speed of the stirrer to 200-300rpm to keep the stirring in a uniform speed state, adding a second buffer solution while stirring until the materials are completely dissolved, keeping the materials clear and transparent, adjusting the pH value to 4.50-6.50 without sediment at the bottom of the liquid preparation tank, and fixing the volume to the final volume;
filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of a microporous filter, storing the filtered solution in a finished product tank, and marking;
step B2: preparation of preserving fluid
Adding part of purified water into a liquid preparation tank on a magnetic stirrer, adjusting the rotating speed of the stirrer to 200-300rpm to keep stirring in a uniform speed state, adding N, N-dihydroxyethyl glycine while stirring until the material is completely dissolved, adding a stabilizer, adding a second preservative after the material is completely dissolved, keeping the solution clear and transparent, adjusting the pH value to 6.30-8.50 without sediment at the bottom of the liquid preparation tank, and fixing the volume to the final volume;
filtering the dissolved preparation solution through a microporous filter membrane according to the operation rules of the microporous filter, and storing the filtered solution for later use for marking;
step B3: preparation of latex microsphere antibody conjugates
Diluting latex microspheres to the concentration of 0.3-3.0% by using a reaction solution, combining the latex microspheres with streptavidin according to a certain proportion, diluting an alpha 1-acid glycoprotein monoclonal antibody to the concentration of 0.05-3.0g/L by using the reaction solution, dialyzing the mixture with biotin according to a certain proportion overnight, adding the mixture into microspheres containing the streptavidin, uniformly mixing, dissolving 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride into the concentration of 0.005-0.3g/L by using the reaction solution, shaking uniformly, dropwise adding the mixture into the mixed solution while reacting for 120 minutes at room temperature, adding a stabilizing agent for sealing, oscillating and uniformly mixing, reacting for 60 minutes at room temperature, centrifuging the mixed solution for 30 minutes at the rotating speed of 14000rpm, sucking off a supernatant, adding a preserving solution, carrying out ultrasonic resuspension, repeatedly centrifuging and cleaning for 3 times, finally centrifuging once to remove the supernatant, adding preservation solution, carrying out ultrasonic resuspension, filling the prepared R2 into a finished product tank, and fixing the volume to the final concentration for marking.
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| CN114137229A (en) * | 2021-12-03 | 2022-03-04 | 苏州普瑞斯生物科技有限公司 | Adiponectin detection reagent production process adopting latex enhanced immunoturbidimetry |
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