CN113791223A - Immunoturbidimetric kit for detecting alpha 1-antitrypsin and preparation method thereof - Google Patents
Immunoturbidimetric kit for detecting alpha 1-antitrypsin and preparation method thereof Download PDFInfo
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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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
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
- G01N33/539—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody involving precipitating reagent, e.g. ammonium sulfate
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/81—Protease inhibitors
- G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- G01N2333/811—Serine protease (E.C. 3.4.21) inhibitors
- G01N2333/8121—Serpins
- G01N2333/8125—Alpha-1-antitrypsin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/70—Mechanisms involved in disease identification
- G01N2800/7095—Inflammation
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Abstract
The invention relates to the field of medical immunity, in particular to an immunoturbidimetric kit for detecting alpha 1-antitrypsin and a preparation method thereof. The kit comprises an R1 reagent, an R2 reagent and a calibrator; the R1 reagent includes: NaCl, EDTA, PEG8000, preservative, buffering agent and water; the R2 reagent includes: anti-alpha 1-antitrypsin antibody, NaCl, preservative, buffer and water. The kit has the advantages of good stability, strong anti-interference capability, long storage period, high detection result accuracy, wide linear detection range and easy clinical popularization.
Description
Technical Field
The invention relates to the field of medical immunity, in particular to an immunoturbidimetric kit for detecting alpha 1-antitrypsin and a preparation method thereof.
Background
Alpha 1-antitrypsin (alpha-1-antiprysin, alpha-1-AT, AAT), also known as alpha-1-protease inhibitor (alpha 1-protease inhibitor, alpha 1-PI), is a single-chain glycoprotein consisting of 394 amino acids, contains 10% -20% of sugars, and is mainly synthesized by the liver. Alpha 1-antitrypsin is a member of the serine protease inhibitor superfamily, is a protease inhibitor, has a biological effect of inhibiting trypsin, chymotrypsin, hyaluronidase, plasmin, elastase and the like, has an inhibitory effect on thrombin, urokinase and other enzymes, is a broad-spectrum protease inhibitor, and is also called alpha 1-protease inhibitor (A1PI) or alpha 1-antitrypsin (A1AP) because it can inhibit various proteases (not only trypsin). Alpha 1-antitrypsin is also an acute phase reaction protein, and in inflammatory diseases, the alpha 1-antitrypsin can enter tissue fluid through capillaries, is often very high in concentration at local inflammation and has a certain limiting effect on acute inflammatory diseases.
The content increase is commonly seen in infectious diseases (bacterial and viral), acute hepatitis, liver cirrhosis, collagen diseases, pregnancy, brain trauma, postoperative surgery, systemic lupus erythematosus, drugs (estrogen, oral contraceptive, adrenal steroid, prostaglandin, etc.), typhus, burn convalescent period, etc. The content reduction is mainly manifested by AAT deficiency, neonatal respiratory distress syndrome, severe hepatitis, acute pancreatitis, emphysema, duodenal bulbar ulcer, nephrotic syndrome, protein-deprivation gastrointestinal disease, hyperthyroidism, disseminated intravascular coagulation, malnutrition, rejection reaction in early stage of immature kidney transplantation, etc.
Currently, the main methods for detecting alpha 1-antitrypsin are: serum trypsin inhibition capacity test, one-way immunodiffusion, rocket immunoelectrophoresis and enzyme linked immunosorbent assay. Wherein, the serum trypsin inhibition capacity test exists: the operation process is complex, the interference of non-antitrypsin in serum is easy to occur, the result is not stable enough, and the method is not suitable for developing a clinical detection kit; the analysis result of the one-way immunodiffusion method is long in time consumption, and the specificity and the accuracy are not high; rocket immunoelectrophoresis has poor accuracy and specificity, and requires special electrophoresis equipment, so the application is rare; the operation process of the enzyme-linked immunosorbent assay is complex and needs much time.
The immunoturbidimetry has the advantages of simple, rapid and sensitive operation and rapid high-throughput measurement by adopting an automatic biochemical analyzer, so that the development of a serum alpha 1-antitrypsin quantitative detection reagent suitable for clinical application in large, medium and small hospitals and basic units is beneficial to disease diagnosis, treatment and prognosis.
Disclosure of Invention
In view of this, the invention provides an immunoturbidimetric kit for detecting alpha 1-antitrypsin and a preparation method thereof. The kit has the advantages of good stability, strong anti-interference capability, long storage period, high detection result accuracy, wide linear detection range and easy clinical popularization.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an immunoturbidimetric kit for detecting alpha 1-antitrypsin, which comprises an R1 reagent, an R2 reagent and a calibrator;
the R1 reagent includes: NaCl, EDTA, PEG8000, preservative, buffering agent and water;
the R2 reagent includes: anti-alpha 1-antitrypsin antibody, NaCl, preservative, buffer and water.
Preferably, the concentration of each component in the R1 reagent is as follows:
preferably, the concentrations of the components in the R1 reagent are:
preferably, the concentration of each component in the R2 reagent is as follows:
preferably, the concentrations of the components in the R2 reagent are:
in the present invention, the buffer is one of phosphate buffer, phosphate-citric acid buffer, MOPS buffer, HEPES buffer.
Preferably, the pH value of the R1 reagent or the R2 reagent is 6.8 to 7.8.
Preferably, the preservative is PC 300.
Preferably, the calibrator is an α 1-antitrypsin solution of known concentration.
Preferably, the concentration of the alpha 1-antitrypsin solution in the calibrator is 0 to 800 mg/dL.
Preferably, the anti- α 1-antitrypsin antibody is an anti-human α 1-antitrypsin antibody.
The invention also provides a preparation method of the immunoturbidimetric kit, which comprises the following steps:
dissolving NaCl, EDTA, PEG8000, preservative and buffering agent in water, adjusting pH value, filtering to obtain R1 reagent;
dissolving an anti-alpha 1-antitrypsin antibody, NaCl, a preservative and a buffering agent in water, adjusting the pH value, and filtering to obtain an R2 reagent;
the R1 reagent, R2 reagent, and calibrator were assembled into a kit.
The invention provides an immunoturbidimetric kit for detecting alpha 1-antitrypsin and a preparation method thereof. The kit comprises an R1 reagent, an R2 reagent and a calibrator; the R1 reagent includes: NaCl, EDTA, PEG8000, preservative, buffering agent and water; the R2 reagent includes: anti-alpha 1-antitrypsin antibody, NaCl, preservative, buffer and water. The invention has the beneficial effects that:
1) the invention adopts an immunoturbidimetry method, and improves the accuracy of reagent measurement value by optimizing a reaction system; the R1 reagent adopts two stabilizers, the proportion of each stabilizer is optimized, and the stability of the reagent is obviously improved.
2) The preferred NaCl content of the stabilizer significantly expands the linear range of the reagent.
3) The preferred EDTA content, a stabilizer, significantly enhances the precision of the reagent.
The invention adopts the innovation points as follows:
1) the linear range of the reagent is remarkably improved by optimizing the content ratio of the stabilizing agent NaCl to the coagulant increasing agent PEG 8000.
2) Optimizing the pH of the R1/R2 reagent and the amount of EDTA used as a stabilizer significantly improves the accuracy of the reagent.
Drawings
FIG. 1 is a graph showing the correlation between the results of the detection using the detection kit of the present invention and the results of the detection using the control kit;
FIG. 2 is a linear relationship diagram of the AAT concentration detection value and the theoretical value of the kit of the invention.
Detailed Description
The invention discloses an immunoturbidimetric kit for detecting alpha 1-antitrypsin and a preparation method thereof, and a person skilled in the art can realize the detection by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The reagents or apparatus used in the present invention are commercially available.
The invention is further illustrated by the following examples:
example 1 kit according to the invention
Composition of kit
1) The component content of the R1 reagent is as follows:
50mM of buffer solution with pH 7.4; 50g/L of stabilizer NaCl; 1.8g/L of EDTA serving as a stabilizer; 60g/L of coagulant aid; 1.0g/L of preservative;
2) the component content of the R2 reagent is as follows:
50mM of buffer solution with pH 7.4; anti-human alpha 1-antitrypsin antibody 600 g/L; 10g/L of stabilizing agent NaCl; preservative 1.0 g/L.
Wherein the buffer solution in the R1 is phosphate buffer solution, the coagulant is PEG8000, and the preservative is PC 300. The buffer solution in R2 is phosphate buffer solution, and the preservative is PC 300.
Preparation method of kit
The reagents R1 and R2 were prepared as follows:
preparation of R1 reagent:
weighing 5.8g of Na according to the formula2HPO4·12H2O、0.593g NaH2PO4·2H2Placing O, 50g of NaCl, 60g of PEG8000, 1.8g of EDTA and 1.0g of PC300 in a clean container, adding deionized water, stirring for 30min to fully dissolve, and adjusting the pH value to 7.4; adding deionized water to constant volume of 1L, and filtering to obtain R1。
Preparation of R2 reagent:
weighing 5.8g of Na according to the formula2HPO4·12H2O、0.593g NaH2PO4·2H2Placing O, 10g NaCl, 1.0g PC300 and 600g antihuman alpha 1-antitrypsin antibody in a clean container, adding deionized water, stirring for 30min to fully dissolve, and adjusting pH to 7.4; adding deionized water to a constant volume of 1L, and filtering to obtain R2.
Third, using method of kit
The detection method of the alpha 1-antitrypsin detection kit described in this example is to use an automatic analyzer of Toshiba toshiba 120, and the operation is as follows: adding deionized water, a sample or a calibrator to 2 mu L, then adding 150 mu L of R1 reagent, mixing uniformly, keeping the temperature at 37 ℃ for 5min, reading the absorbance A1 relative to the blank, then adding 50 mu L of R2 reagent, mixing uniformly, keeping the temperature at 37 ℃ for 5min, reading the absorbance A2, and calculating the delta A (A) 2-A1.
Example 2 kit according to the invention
Composition of kit
1) The component content of the R1 reagent is as follows:
50mM of buffer solution with pH 6.8; 60g/L of a stabilizer NaCl; 1.8g/L of EDTA serving as a stabilizer; 65g/L of coagulant increasing agent; 1.0g/L of preservative;
2) the component content of the R2 reagent is as follows:
50mM of buffer solution with the pH value of 6.8; 500g/L of anti-human alpha 1-antitrypsin antibody; 10g/L of stabilizing agent NaCl; preservative 1.0 g/L.
Wherein the buffer solution in the R1 is phosphate buffer solution, the coagulant is PEG8000, and the preservative is PC 300. The buffer solution in R2 is phosphate buffer solution, and the preservative is PC 300.
Preparation method of kit
The reagents R1 and R2 were prepared as follows:
preparation of R1 reagent:
weighing 5.8g of Na according to the formula2HPO4·12H2O、0.593g NaH2PO4·2H2Adding O, 60g NaCl, 65g PEG8000, 1.8g EDTA, 1.0g PC300 into clean container, adding deionized water, stirring for 30min to make it fullyDissolving and adjusting the pH value to 6.8; adding deionized water to a constant volume of 1L, and filtering to obtain R1.
Preparation of R2 reagent:
weighing 5.8g of Na according to the formula2HPO4·12H2O、0.593g NaH2PO4·2H2Placing O, 10g NaCl, 1.0g PC300 and 500g antihuman alpha 1-antitrypsin antibody in a clean container, adding deionized water, stirring for 30min to fully dissolve, and adjusting pH to 6.8; adding deionized water to a constant volume of 1L, and filtering to obtain R2.
Third, using method of kit
The same as in example 1.
Example 3 kit according to the invention
Composition of kit
1) The component content of the R1 reagent is as follows:
50mM of buffer solution with pH 7.8; 45g/L of a stabilizer NaCl; 2.0g/L of EDTA as a stabilizer; 55g/L of coagulant aid; 1.0g/L of preservative;
2) the component content of the R2 reagent is as follows:
50mM of buffer solution with pH 7.8; 550g/L of anti-human alpha 1-antitrypsin antibody; 10g/L of stabilizing agent NaCl; preservative 1.0 g/L.
Wherein the buffer solution in the R1 is phosphate buffer solution, the coagulant is PEG8000, and the preservative is PC 300. The buffer solution in R2 is phosphate buffer solution, and the preservative is PC 300.
Preparation method of kit
The reagents R1 and R2 were prepared as follows:
preparation of R1 reagent:
weighing 5.8g of Na according to the formula2HPO4·12H2O、0.593g NaH2PO4·2H2Placing O, 45g of NaCl, 55g of PEG8000, 2.0g of EDTA and 1.0g of PC300 in a clean container, adding deionized water, stirring for 30min to fully dissolve, and adjusting the pH value to 7.8; adding deionized water to a constant volume of 1L, and filtering to obtain R1.
Preparation of R2 reagent:
weighing 5.8g of Na according to the formula2HPO4·12H2O、0.593g NaH2PO4·2H2Placing O, 10g NaCl, 1.0g PC300 and 550g anti-human alpha 1-antitrypsin antibody in a clean container, adding deionized water, stirring for 30min to fully dissolve, and adjusting pH to 7.8; adding deionized water to a constant volume of 1L, and filtering to obtain R2.
Third, using method of kit
The same as in example 1.
Example 4 kits according to the invention
Composition of kit
1) The component content of the R1 reagent is as follows:
20mM of buffer solution with pH 7.4; 50g/L of stabilizer NaCl; 1.8g/L of EDTA serving as a stabilizer; 60g/L of coagulant aid; 2.0g/L of preservative;
2) the component content of the R2 reagent is as follows:
pH7.4 buffer 20 mM; 500g/L of anti-human alpha 1-antitrypsin antibody; 15g/L of a stabilizer NaCl; preservative 2.0 g/L.
Wherein the buffer solution in R1 is MOPS buffer solution, the coagulant is PEG8000, and the preservative is PC 300. The buffer in R2 is MOPS buffer, and the preservative is PC 300.
Preparation method of kit
The reagents R1 and R2 were prepared as follows:
preparation of R1 reagent:
weighing 4.186g MOPS, 50g NaCl, 60g PEG8000, 1.8g EDTA and 2.0g PC300 according to the formula, adding deionized water, stirring for 30min to fully dissolve, and adjusting pH to 7.4; adding deionized water to a constant volume of 1L, and filtering to obtain R1.
Preparation of R2 reagent:
4.186g of MOPS, 15g of NaCl, 2.0g of PC300 and 500g of anti-human alpha 1-antitrypsin antibody are weighed according to the formula and put in a clean container, deionized water is added and stirred for 30min to fully dissolve the antibodies, and the pH value is adjusted to 7.4; adding deionized water to a constant volume of 1L, and filtering to obtain R2.
Third, using method of kit
The same as in example 1.
Example 5 kits according to the invention
Composition of kit
1) The component content of the R1 reagent is as follows:
50mM of buffer solution with pH 7.4; 45g/L of a stabilizer NaCl; 1.0g/L of EDTA serving as a stabilizer; 55g/L of coagulant aid; 1.0g/L of preservative;
2) the component content of the R2 reagent is as follows:
50mM of buffer solution with pH 7.4; 650g/L of anti-human alpha 1-antitrypsin antibody; 10g/L of stabilizing agent NaCl; preservative 1.0 g/L.
Wherein the buffer solution in R1 is HEPES buffer solution, the coagulant is PEG8000, and the preservative is PC 300. The buffer in R2 is HEPES buffer, and the preservative is PC 300.
Preparation method of kit
The reagents R1 and R2 were prepared as follows:
preparation of R1 reagent:
weighing 11.91g of HEPES, 45g of NaCl, 55g of PEG8000, 1.0g of EDTA and 1.0g of PC300 according to the formula, adding deionized water, stirring for 30min to fully dissolve the materials, and adjusting the pH value to 7.4; adding deionized water to a constant volume of 1L, and filtering to obtain R1.
Preparation of R2 reagent:
weighing 11.91g of HEPES, 10g of NaCl, 1.0g of PC300 and 650g of anti-human alpha 1-antitrypsin antibody according to the formula, adding deionized water, stirring for 30min to fully dissolve the antibodies, and adjusting the pH value to 7.4; adding deionized water to a constant volume of 1L, and filtering to obtain R2.
Third, using method of kit
The same as in example 1.
Comparative example 1
An alpha 1-antitrypsin detection kit comprising R1 reagent and R2 reagent, in accordance with example 1, except that DETA is used in a larger amount. The R1 reagent comprises the following components: 5.8g Na2HPO4·12H2O、0.593g NaH2PO4·2H2O、50g NaCl、60g PEG8000、3.0g EDTA、1.0g PC300；
The preparation method of the R1 reagent comprises the following steps:
weighing Na according to the required amount2HPO4·12H2O、NaH2PO4·2H2O, sodium chloride, polyethylene glycol 8000, EDTA and a preservative Proclin300, and the pH value is adjusted after the materials are put into a stirrer and stirred uniformly; adding deionized water to 1L, stirring uniformly, and filtering to obtain R1.
Comparative example 2
An α 1-antitrypsin detection kit comprising R1 reagent and R2 reagent, in accordance with example 1, except that the pH of R1 reagent and R2 reagent are different. The R1 reagent comprises the following components: 5.8g Na2HPO4·12H2O、0.593g NaH2PO4·2H2O、50g NaCl、60g PEG8000、1.8g EDTA、1.0g PC300，pH6.0；
The preparation method of the R1 reagent comprises the following steps:
weighing Na according to the required amount2HPO4·12H2O、NaH2PO4·2H2O, sodium chloride, polyethylene glycol 8000, EDTA and a preservative Proclin300, and the pH value is adjusted to 6.0 after the materials are put into a stirrer to be uniformly stirred; adding deionized water to 1L, stirring uniformly, and filtering to obtain R1.
The pH of the R2 reagent was 6.0.
Comparative example 3
An α 1-antitrypsin detection kit comprising R1 reagent and R2 reagent, in accordance with example 1, except that the pH of R1 reagent and R2 reagent are different. The R1 reagent comprises the following components: 5.8g Na2HPO4·12H2O、0.593g NaH2PO4·2H2O、50g NaCl、60g PEG8000、1.8g EDTA、1.0g PC300，pH8.5；
The preparation method of the R1 reagent comprises the following steps:
weighing Na according to the required amount2HPO4·12H2O、NaH2PO4·2H2O, sodium chloride, polyethylene glycol 8000, EDTA and a preservative Proclin300, and the pH value is adjusted to 6.0 after the materials are put into a stirrer to be uniformly stirred; adding deionized water to 1L, stirring uniformly, and filtering to obtain R1.
The pH of the R2 reagent was 8.5.
Comparative example 4
An α 1-antitrypsin detection kit comprising R1 reagent and R2 reagent, in accordance with example 1, except that NaCl is used in a smaller amount. The R1 reagent comprises the following components: 5.8g Na2HPO4·12H2O、0.593g NaH2PO4·2H2O、30g NaCl、60g PEG8000、1.8gEDTA、1.0g PC300；
The preparation method of the R1 reagent comprises the following steps:
weighing Na according to the required amount2HPO4·12H2O、NaH2PO4·2H2O, sodium chloride, polyethylene glycol 8000, EDTA and a preservative Proclin300, and the pH value is adjusted after the materials are put into a stirrer and stirred uniformly; adding deionized water to 1L, stirring uniformly, and filtering to obtain R1.
Comparative example 5
An alpha 1-antitrypsin detection kit comprising R1 reagent and R2 reagent, in accordance with example 1, except that NaCl, PEG8000, are used in small amounts. The R1 reagent comprises the following components: 5.8g Na2HPO4·12H2O、0.593g NaH2PO4·2H2O、45g NaCl、40g PEG8000、1.8gEDTA、1.0g PC300；
The preparation method of the R1 reagent comprises the following steps:
weighing Na according to the required amount2HPO4·12H2O、NaH2PO4·2H2O, sodium chloride, polyethylene glycol 8000, EDTA and a preservative Proclin300, and the pH value is adjusted after the materials are put into a stirrer and stirred uniformly; adding deionized water to 1L, stirring uniformly, and filtering to obtain R1.
Comparative example 6
An alpha 1-antitrypsin detection kit comprising R1 reagent and R2 reagent, in accordance with example 1, except that PEG8000 is used in a smaller amount. The R1 reagent comprises the following components: 5.8g Na2HPO4·12H2O、0.593g NaH2PO4·2H2O、50g NaCl、35g PEG8000、1.8gEDTA、1.0g PC300；
The preparation method of the R1 reagent comprises the following steps:
weighing Na according to the required amount2HPO4·12H2O、NaH2PO4·2H2O, sodium chloride, polyethylene glycol 8000, EDTA and a preservative Proclin300, and the pH value is adjusted after the materials are put into a stirrer and stirred uniformly; adding deionized water to 1L, stirring uniformly, and filtering to obtain R1.
Comparative example 7
An alpha 1-antitrypsin detection kit comprising R1 reagent and R2 reagent, in accordance with example 1, except without EDTA. The R1 reagent comprises the following components: 5.8g Na2HPO4·12H2O、0.593g NaH2PO4·2H2O、50g NaCl、60g PEG8000、1.0g PC300；
The preparation method of the R1 reagent comprises the following steps:
weighing Na according to the required amount2HPO4·12H2O、NaH2PO4·2H2O, sodium chloride, polyethylene glycol 8000 and a preservative Proclin300, and adjusting the pH value after uniformly stirring in a stirrer; adding deionized water to 1L, stirring uniformly, and filtering to obtain R1.
Comparative example 8
An alpha 1-antitrypsin detection kit comprising R1 reagent and R2 reagent, in accordance with example 1, except that EDTA is used in a smaller amount. The R1 reagent comprises the following components: 5.8g Na2HPO4·12H2O、0.593g NaH2PO4·2H2O、50g NaCl、60g PEG8000、0.5g EDTA、1.0g PC300；
The preparation method of the R1 reagent comprises the following steps:
weighing Na according to the required amount2HPO4·12H2O、NaH2PO4·2H2O, sodium chloride, polyethylene glycol 8000, EDTA and a preservative Proclin300, and the pH value is adjusted after the materials are put into a stirrer and stirred uniformly; adding deionized water to 1L, stirring uniformly, and filtering to obtain R1.
Comparative example 9
The reagent R1 and the reagent R2 are prepared according to the formula disclosed in patent example 3 with publication number CN 111781372A.
The R1 reagent comprises the following components in percentage by weight: HEPES 23.83g/L, NaOH 0.4g/L, NaCl 9.0g/L, EDTA-Na21.1g/L, PEG-600015 g/L, PEG-80008 g/L, Tween 200.5%, NaN30.01g/L, pH6.4, and the solvent is purified water.
The R2 reagent comprises the following components in percentage by weight: NaH2PO4·2H2O 1.48g/L，Na2HPO4·12H2O 9.71g/L，NaCl 9.0g/L，EDTA-Na21.1g/L, BSA 2.4g/L, anti-human alpha 1-antitrypsin antibody 60%, NaN30.01g/L, pH7.0, and the solvent is purified water.
Comparative example 10
The reagent R1 and the reagent R2 were formulated in patent example 1 with publication number CN 112285356A.
The R1 reagent includes: 1.6g/L NaH2PO4·2H2O，8.75g/L Na2HPO4·12H2O，8.0g/L NaCl，60g/L PEG-6000，0.8mL/L Proclin-950，pH 7.0。
The R2 reagent includes: 1.6g/L NaH2PO4·2H2O，8.75g/L Na2HPO4·12H2O, 8.0g/L NaCl, 1g/L BSA, anti-human alpha 1-antitrypsin antibody 60%, 0.8mL/L Proclin-950, pH 7.0.
Comparative example 11
Refer to the formulation in patent example 1 with publication number CN 112285356A to prepare R1 reagent and R2 reagent, and adjust the pH of R1 reagent to 8.0.
The R1 reagent includes: 1.6g/L NaH2PO4·2H2O，8.75g/L Na2HPO4·12H2O，8.0g/L NaCl，60g/L PEG-6000，0.8mL/L Proclin-950，pH 8.0。
The R2 reagent includes: 1.6g/L NaH2PO4·2H2O，8.75g/L Na2HPO4·12H2O, 8.0g/L NaCl, 1g/L BSA, anti-human alpha 1-antitrypsin antibody 60%, 0.8mL/L Proclin-950, pH 7.0.
Examples of test effects
The performance evaluation result levels of the kits disclosed in the embodiments 1-5 of the invention are basically consistent, and the kits prepared in the embodiments 1-5 are taken as examples to verify the correlation performance such as correlation, linear range, precision, anti-interference performance, stability and the like.
(1) Standard curve formulation
The reagent of the implementation method is tested by a Toshiba 120FR full-automatic biochemical analyzer, the testing wavelength is 600nm, 2 mu L of sample or calibrator is taken, 150 mu L of R1 reagent is added, the temperature is kept at 37 ℃ for 5min, and the absorbance A1 is read relative to the blank; then adding 50 mu L R2 reagent, incubating for 5min at 37 ℃, and reading the absorbance A2, wherein the reaction absorbance delta A is A2-A1; and performing multi-point calibration by using the standard substance, calculating by using a spline function to obtain a calibration curve, and searching the AAT concentration value in the sample from the working curve.
(2) Correlation experiments
A commercially approved alpha 1-antitrypsin kit with excellent accuracy is used as a control group, the kits of the examples are used as experimental groups for comparison experiments, and 40 samples are detected, wherein the detection results are shown in Table 1. Taking the test result of a contrast kit on the market as an independent variable of a horizontal coordinate, taking the test result of the kit of the invention as a dependent variable of a vertical coordinate, and making a linear regression curve to obtain:
example 1 the regression equation is Y0.9836X +2.9251 and the linear correlation coefficient R0.9986;
example 2 regression equation Y0.9675X +2.0003, linear correlation coefficient R0.9977;
example 3 regression equation Y is 0.9527X +1.1219 and linear correlation coefficient R is 0.9985;
example 4 regression equation Y0.9737X +4.3591, linear correlation coefficient R0.9981;
example 5 regression equation Y is 0.9655X +4.0106, linear correlation coefficient R is 0.9975;
as can be seen, the examples 1-5 have good linear relationship and the test results can be effectively used as clinical tests. Example 1 the correlation curve is shown in figure 1.
Comparative examples 1-3 the results are shown in Table 2;
comparative example 1 the regression equation is Y0.9051X +13.744 and the linear correlation coefficient R is 0.956;
comparative example 2 the regression equation is Y1.1389X +11.353 and the linear correlation coefficient R is 0.959;
comparative example 3 the regression equation is Y0.8609X +9.3461 and the linear correlation coefficient R0.9554;
the results of tables 1-2 show that: optimizing the pH of the R1/R2 reagent and the stabilizer EDTA significantly improved the accuracy of the reagent.
TABLE 1 EXAMPLES 1-5 correlation test results (unit: mg/dL)
TABLE 2 results of correlation test (unit: mg/dL) for comparative examples 1 to 3
(3) Linear experiment
The samples were mixed (aliquoted) into 11 dilution concentration samples and averaged 2 times per sample. Diluting the high-value sample by a certain multiple to be used as a low-value sample of 8mg/dL, and adding the high-value sample of 800mg/dL into the mixed normal sample to be used as a linear high value; the high and low value samples are mixed in proportion into 11 arithmetic dilution samples. The results of the linearity measurements are shown in Table 3. And (3) solving a linear regression equation by taking the theoretical AAT concentration as an independent variable X of an abscissa and taking an actual test value as a dependent variable Y of an ordinate, calculating a linear regression correlation coefficient r, and displaying a result:
example 1 the linear regression equation is Y0.9852X-4.1997 with a correlation coefficient r of 0.9994;
example 2 the linear regression equation is Y-0.9882X-9.1327 with correlation coefficient r-0.9992;
example 3 the linear regression equation is Y-0.9865X-9.4371 with correlation coefficient r-0.9992;
example 4 the linear regression equation is Y-0.9835X-7.5034 with correlation coefficient r-0.9991;
example 5 the linear regression equation is Y0.9932X-10.537 with a correlation coefficient r of 0.9989;
the invention shows that the invention has better relativity in the linear range of 8 mg/dL-800 mg/dL. Example 1 the linear range curve is shown in figure 2.
Comparative examples 4-6 the results are shown in Table 4;
comparative example 4 the linear regression equation is Y0.7955X +26.909 with a correlation coefficient r of 0.987;
comparative example 5 the linear regression equation is Y0.7979X +23.935 with correlation coefficient r 0.9891;
comparative example 6 the linear regression equation is Y0.8023X +26.945 with correlation coefficient r 0.9891;
tables 3-4 the results show that: the content ratio of the stable NaCl to the coagulant PEG8000 is optimized, and the linear range of the reagent is remarkably improved.
Comparative examples 9 to 11 the results are shown in Table 5;
comparative example 9 the linear regression equation was 0.2908X +135.77 with a correlation coefficient r of 0.2454;
comparative example 10 the linear regression equation is Y0.8476X +21.706 with correlation coefficient r 0.9815;
comparative example 11 the linear regression equation is Y0.8453X +23.138 with a correlation coefficient r of 0.985;
example 1 the linear regression equation is Y0.9892X-5.7552 with a correlation coefficient r of 0.9993;
table 5 the results show that: when the pH (6.8-7.8) range of the reagent is exceeded, the linear range of the reagent is reduced by replacing the content ratio of NaCl to the coagulant PEG8000, and the accuracy of measuring a high-value sample of the reagent is influenced.
TABLE 3 results of linear range analysis of examples 1-5
TABLE 4 results of linear range analysis of comparative examples 4 to 6
TABLE 5 results of linear range analysis of comparative examples 9 to 11
(4) Detection of precision
Two serum samples with high and low AAT concentration are respectively taken, each serum sample is continuously tested 20 times, the variation coefficient is calculated, the precision results of the examples are shown in table 6, and the precision results of the comparative examples 1 and 7-8 are shown in table 7. As can be seen from the experimental results in tables 6-7, the precision of the reagent can be significantly improved by adding the EDTA as the stabilizer into the R1 reagent, and the effect of improving the precision of the reagent is reduced after the content of the EDTA exceeds a certain concentration.
TABLE 6 results of the precision measurements of examples 1 to 5
TABLE 7 results of precision measurements of comparative examples 1, 7-8
(5) Anti-interference detection
Dividing the serum of a clinical normal patient into two parts, adding the interfering substance with the highest concentration into one part, adding the solvent with the same amount into the other part, carrying out 3-gradient equal-difference dilution on the samples with and without the interfering substance, detecting each sample for three times, reversing the detection sequence, and calculating the deviation of the measured value. The interference rejection of the examples was evaluated and the results are shown in Table 8.
TABLE 8 results of anti-interference tests in examples 1-5
(6) Stability detection
The kit of the invention was tested for long term stability. The kit is taken and put on a testing instrument for calibration, the kit is stored for 18 months in a sealed manner at the temperature of 2-8 ℃, long-term stability testing is carried out on serum samples with the concentrations of 130mg/dL and 240mg/dL respectively in3 months, 6 months, 9 months, 12 months and 18 months, and the deviation value of the testing result after 18 months of bottle opening is calculated, and the result is shown in table 9.
TABLE 9 stability test results of examples 1-5
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. An immunoturbidimetric kit for detecting alpha 1-antitrypsin, which is characterized by comprising an R1 reagent, an R2 reagent and a calibrator;
the R1 reagent includes: NaCl, EDTA, PEG8000, preservative, buffering agent and water;
the R2 reagent includes: anti-alpha 1-antitrypsin antibody, NaCl, preservative, buffer and water.
2. The immunoturbidimetric kit according to claim 1, wherein the concentrations of the components in the R1 reagent are as follows:
3. the immunoturbidimetric kit according to claim 1, wherein the concentrations of the components in the R2 reagent are as follows:
4. the immunoturbidimetric kit of claim 1, wherein the buffer is one of phosphate buffer, phosphate-citrate buffer, MOPS buffer, HEPES buffer.
5. The immunoturbidimetric kit of claim 4, wherein the pH of the R1 reagent or the R2 reagent is 6.8 to 7.8.
6. The immunoturbidimetric kit of claim 1, wherein the preservative is PC 300.
7. The immunoturbidimetric kit of claim 1, wherein the calibrator is an α 1-antitrypsin solution of known concentration.
8. The immunoturbidimetric kit of claim 7, wherein the concentration of the α 1-antitrypsin solution in the calibrator is 0 to 800 mg/dL.
9. The immunoturbidimetric kit of any one of claims 1 to 8, wherein the anti- α 1-antitrypsin antibody is an anti-human α 1-antitrypsin antibody.
10. The method for preparing an immunoturbidimetric kit according to any of claims 1 to 9, comprising the steps of:
dissolving NaCl, EDTA, PEG8000, preservative and buffering agent in water, adjusting pH value, filtering to obtain R1 reagent;
dissolving an anti-alpha 1-antitrypsin antibody, NaCl, a preservative and a buffering agent in water, adjusting the pH value, and filtering to obtain an R2 reagent;
the R1 reagent, R2 reagent, and calibrator were assembled into a kit.
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| CN112285356A (en) * | 2019-07-25 | 2021-01-29 | 苏州普瑞斯生物科技有限公司 | Preparation method of alpha 1-antitrypsin immunoturbidimetry detection kit |
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| KR910011281A (en) * | 1989-12-21 | 1991-08-07 | 강현영 | Separation and Purification of Alpha-1-antitrypsin from Serum and Assay Kits of Alpha-1-antitrypsin in Blood |
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