CN111122867A - Pepsinogen I detection kit and preparation method thereof - Google Patents
Pepsinogen I detection kit and preparation method thereof Download PDFInfo
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- CN111122867A CN111122867A CN201911287921.XA CN201911287921A CN111122867A CN 111122867 A CN111122867 A CN 111122867A CN 201911287921 A CN201911287921 A CN 201911287921A CN 111122867 A CN111122867 A CN 111122867A
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- G—PHYSICS
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- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- 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
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- G01—MEASURING; TESTING
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- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
- G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
- G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
- G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
- G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
- G01N2333/96472—Aspartic endopeptidases (3.4.23)
- G01N2333/96475—Aspartic endopeptidases (3.4.23) with definite EC number
- G01N2333/96477—Pepsin (3.4.23.1; 3.4.23.2; 3.4.23.3)
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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
- G01N2800/06—Gastro-intestinal diseases
- G01N2800/062—Gastritis or peptic ulcer disease
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Abstract
The invention discloses a pepsinogen I detection kit, which comprises an R1 reagent and an R2 reagent, wherein the R1 reagent comprises 50-150mmol/L of sodium phosphate buffer solution, 2-3g/L of bovine serum albumin, 2-3% of methylisothiazolinone, 1.0-5.0g/L of condensation product of fatty alcohol and ethylene oxide, 3-5mmol/L of magnesium chloride, 20-30mmol/L of ammonium formate and the balance of deionized water; the R2 reagent comprises 1.3-2mg/ml pepsinogen I antibody emulsion solution, 2-3g/L bovine serum albumin, 2-3% methylisothiazolinone in concentration and the balance of deionized water; the pepsinogen I detection kit and the preparation method thereof are prepared, and the pepsinogen I detection kit can effectively solve the chyle problem, has strong anti-interference capability and has the effects of high stability and high sensitivity.
Description
Technical Field
The invention relates to a pepsinogen I detection kit and a preparation method thereof.
Background
Pepsinogen (pepsinogen), synthesized by the main cells of the secretory gland, is reacted in the stomach cavity by hydrochloric acid (HCL) or already active pepsin (pepsin) to become pepsin, which decomposes protein into fat, peptone and a small amount of polypeptide, and the serum pepsinogen level reflects the form and function of the gastric mucosa at different sites: PGI is a pointer for detecting the function of the cells of the gastric acid gland, and the PGI is increased due to the increase of gastric acid secretion, and the PGI is reduced due to the reduction of secretion or the atrophy of gastric mucosa glands; PGII is associated with greater lesion of the gastric fundus mucosa (relative to the antral mucosa), and its elevation is associated with atrophy of the gastric fundus ducts, metaplasia of the gastric epithelium or metaplasia, abnormal proliferation; the progressive reduction of the PGI/II ratio is associated with the progression of gastric mucosal atrophy, and the progression of gastric diseases by Pepsinogen (PG) can be generally expressed as: superficial gastritis, gastric mucosa erosion ulcer, atrophic gastritis, gastric cancer and other diseases have good diagnosis and screening effects.
The common latex enhanced immunoturbidimetry method is adopted for the general pepsinogen I, the method is mainly chyle interference, complete statistics is rejected, the ratio of chyle blood is 0.5-1% in blood drawing detection, which is related to modern people who often eat some high-fried and high-calorie foods, but the elimination of the problems in the market is realized by adding a large amount of chyle eliminating agents, although the chyle problem can be further reduced, a large amount of foreign substances are added, which inevitably brings certain influence on detection data, and therefore, a test box which can solve the chyle problem and can not influence the final detection result is needed in the market.
Disclosure of Invention
The invention aims to solve the technical problem of providing a pepsinogen I detection kit which can effectively solve the problem of chyle, has strong anti-interference capability and high stability and sensitivity and a preparation method thereof.
In order to solve the problems, the invention adopts the following technical scheme: a pepsinogen I detection kit comprises an R1 reagent and an R2 reagent, wherein the R1 reagent comprises 50-150mmol/L of sodium phosphate buffer solution, 2-3g/L of bovine serum albumin, 2-3% of methylisothiazolinone, 1.0-5.0g/L of fatty alcohol and ethylene oxide condensate, 3-5mmol/L of magnesium chloride, 20-30mmol/L of ammonium formate and the balance of deionized water; the R2 reagent comprises 1.3-2mg/ml pepsinogen I antibody emulsion solution, 2-3g/L bovine serum albumin, 2-3% methylisothiazolinone in concentration and the balance of deionized water.
The further scheme is as follows: the pepsinogen I antibody emulsion solution comprises a 15% solution of latex particles coated by an anti-human pepsinogen I antibody, wherein the latex particles are monoclonal antibody cross-linked polymer nano microspheres which adopt polystyrene as a raw material, and the monoclonal antibody adopts a mouse or cattle antibody.
The further scheme is as follows: the preparation of the fatty alcohol and ethylene oxide condensation compound comprises the steps of ring-opening polymerization of ethylene oxide in a reaction kettle, condensation with alcohol to generate fatty alcohol-polyoxyethylene ether, wherein the reaction kettle is in a vacuum state, the pressure value is 0.3MPa, the temperature is controlled between 130-plus-material temperature and 150 ℃, the time is 1 hour, and the heat preservation is continued for 2 hours after 1 hour.
The further scheme is as follows: the temperature of the sodium phosphate buffer solution in the R1 reagent and the R2 reagent is 20 +/-1 ℃, and the pH is 8.
The further scheme is as follows: the sodium phosphate buffer solution is prepared by dissolving sodium phosphate in 1L of distilled water, then dropwise adding 1mol/L of KCl for preparation until the pH is 8, diluting with distilled water, storing in an ultraviolet sterilization cabinet for 30min, and storing in a 135 ℃ high-temperature sterilization box for 10 min.
The further scheme is as follows: the volume ratio of the R1 reagent to the R2 reagent is 2: 1.5.
The invention also discloses a preparation method of the pepsinogen I detection kit, which comprises the following steps:
1) adding bovine serum albumin, a condensate of methyl isothiazolinone, fatty alcohol and ethylene oxide, magnesium chloride and ammonium formate into a sodium phosphate buffer solution, carrying out vortex oscillation for 10min, standing at 4-5 ℃ for 2h, centrifuging at 800R/min for 20min, standing for 2h, taking supernatant fluid for later use at 3-5 ℃ to prepare an R1 reagent;
2) firstly, 2-3g/L of bovine serum albumin, 2-3% of methylisothiazolinone and the balance of deionized water are added into pepsinogen I antibody emulsion solution, vortex oscillation is carried out for 20min, ultrasonic crushing is carried out for 10min, and after the mixture is placed for 2h at 4-5 ℃, supernatant is taken and is kept at 3-5 ℃ for later use to prepare R2 reagent;
3) and mixing the R1 reagent and the R2 reagent to obtain the pepsinogen I detection kit.
The invention has the beneficial effects that:
according to the invention, the R1 reagent and the R2 reagent are adopted, the condensation product of fatty alcohol and ethylene oxide is added into the R1 reagent as a surfactant, chyle interference of the reagents can be eliminated, and bovine serum albumin, magnesium chloride and ammonium formate are added into the R1 reagent, so that the stability of the reagents for detecting stomach diseases can be maintained, the stability and the anti-interference capability of the prepared pepsinogen I detection kit are obviously improved, and the kit can be popularized and used in the field of related biochemical reagents.
Detailed Description
The following detailed description of the preferred embodiments of the present invention is provided to enable those skilled in the art to more readily understand the advantages and features of the present invention and to clearly and unequivocally define the scope of the present invention.
Example one
A pepsinogen I detection kit comprises an R1 reagent and an R2 reagent, wherein the R1 reagent comprises a Good's buffer solution 100mmol/L, sodium chloride 80mmol/L, magnesium chloride 12mmol/L, α -glucosidase 3kU/L, an anti-sialidase monoclonal antibody 27mg/L, bovine serum albumin 3G/L, polyvinylpyrrolidone 50mg/L, N-acetylcysteine 50mmol/L, and the balance of distilled water, the R2 reagent comprises a Good's buffer solution 150mmol/L, 4, 6-ethylene- α, D-malto 7 glucoside-p-nitrophenol (EPS-G7)1.6mmol/L, bovine serum albumin 4G/L, ascorbic acid oxidase 1200U/L, and the balance of distilled water.
Reagent R1 reagent and R2 reagent preparation:
1) adding bovine serum albumin, a condensate of methyl isothiazolinone, fatty alcohol and ethylene oxide, magnesium chloride and ammonium formate into a sodium phosphate buffer solution, carrying out vortex oscillation for 10min, standing at 4-5 ℃ for 2h, centrifuging at 800R/min for 20min, standing for 2h, taking supernatant fluid for later use at 3-5 ℃ to prepare an R1 reagent;
2) firstly, 2-3g/L of bovine serum albumin, 2-3% of methylisothiazolinone and the balance of deionized water are added into pepsinogen I antibody emulsion solution, vortex oscillation is carried out for 20min, ultrasonic crushing is carried out for 10min, and after the mixture is placed for 2h at 4-5 ℃, supernatant is taken and is kept at 3-5 ℃ for later use to prepare R2 reagent;
3) and mixing the R1 reagent and the R2 reagent to obtain the pepsinogen I detection kit.
Preparation of fatty alcohol and ethylene oxide condensate: the method comprises the steps of ring-opening polymerization of ethylene oxide in a reaction kettle, condensation with alcohol to generate fatty alcohol-polyoxyethylene ether, wherein the reaction kettle is in a vacuum state, the pressure value is 0.3MPa, the temperature is controlled to be 130 ℃, the time is 1 hour, and the heat preservation is continued for 2 hours after 1 hour.
Effect of different chyle concentrations on serum
The normal value range of the pepsinogen I is 70-200ug/L, the chyle content of a person is specially selected to be increased from 5 percent to 50 percent each time, the chyle content is compared with the common reagents in the market, the detection is repeatedly carried out at an interval of 60S in a continuous monitoring method, and the average value and the deviation are calculated.
Table 1 shows the results of the comparison of the kit with the common detection agent on the market after the adding amount of chyle is increased by 5 percent
As shown in table 1, although the data detected at the two sides are not very different, which indicates that the stability of the two products is good in each detection, the detection effect of the comparison product is obviously different from that of the comparison product which is not added with chyle in the process of adding 5% -50% of chyle, so that the influence of the ratio of the plate product on the elimination of chyle in serum is obviously better than that of the comparison product.
Example two
A pepsinogen I detection kit comprises an R1 reagent and an R2 reagent, wherein the R1 reagent comprises 100mmol/L of sodium phosphate buffer solution, 2g/L of bovine serum albumin, 3% methylisothiazolinone in concentration, 3g/L of fatty alcohol and ethylene oxide condensate, 4mmol/L of magnesium chloride, 25mmol/L of ammonium formate and the balance of deionized water; the R2 reagent comprises pepsinogen I antibody emulsion solution 2mg/ml, bovine serum albumin 2g/L, 3% methylisothiazolinone in concentration, and the balance of deionized water.
Reagent R1 reagent and R2 reagent preparation:
1) adding bovine serum albumin, a condensate of methyl isothiazolinone, fatty alcohol and ethylene oxide, magnesium chloride and ammonium formate into a sodium phosphate buffer solution, carrying out vortex oscillation for 10min, standing at 5 ℃ for 2h, centrifuging at 800R/min for 20min, standing for 2h, taking a supernatant at 5 ℃ for later use, and preparing an R1 reagent;
2) firstly, adding bovine serum albumin, methylisothiazolinone and the balance of deionized water into a pepsinogen I antibody emulsion solution, carrying out vortex oscillation for 20min, carrying out ultrasonic crushing for 10min, standing for 2h at 4-5 ℃, and taking supernatant fluid at 3 ℃ for later use to prepare an R2 reagent;
3) and mixing the R1 reagent and the R2 reagent to obtain the pepsinogen I detection kit.
Preparation of fatty alcohol and ethylene oxide condensate: the method comprises the steps of ring-opening polymerization of ethylene oxide in a reaction kettle, condensation with alcohol to generate fatty alcohol-polyoxyethylene ether, wherein the reaction kettle is in a vacuum state, the pressure value is 0.3MPa, the temperature is controlled to be 130 ℃, the time is 1 hour, and the heat preservation is continued for 2 hours after 1 hour.
Effect of different chyle concentrations on serum
The normal value range of the pepsinogen I is 70-200ug/L, the chyle content of a person is specially selected to be increased from 5 percent to 50 percent each time, the chyle content is compared with the common reagents in the market, the detection is repeatedly carried out at an interval of 60S in a continuous monitoring method, and the average value and the deviation are calculated.
Table 2 shows the results of the comparison of the reagent kit with the common detection agent on the market after the adding amount of chyle is increased by 5 percent
As shown in table 1, although the data detected at the two sides are not very different, which indicates that the stability of the two products is good in each detection, the detection effect of the comparison product is obviously different from that of the comparison product which is not added with chyle in the process of adding 5% -50% of chyle, so that the influence of the ratio of the plate product on the elimination of chyle in serum is obviously better than that of the comparison product.
EXAMPLE III
A pepsinogen I detection kit comprises an R1 reagent and an R2 reagent, wherein the R1 reagent comprises 100mmol/L of sodium phosphate buffer solution, 3g/L of bovine serum albumin, 3g/L of methylisothiazolinone with the concentration of 2%, 3g/L of fatty alcohol and ethylene oxide condensate, 3mmol/L of magnesium chloride, 30mmol/L of ammonium formate and the balance of deionized water; the R2 reagent comprises pepsinogen I antibody emulsion solution 2mg/ml, bovine serum albumin 2g/L, 3% methylisothiazolinone in concentration, and the balance of deionized water.
Reagent R1 reagent and R2 reagent preparation:
1) adding bovine serum albumin, a condensate of methyl isothiazolinone, fatty alcohol and ethylene oxide, magnesium chloride and ammonium formate into a sodium phosphate buffer solution, carrying out vortex oscillation for 10min, standing at 5 ℃ for 2h, centrifuging at 800R/min for 20min, standing for 2h, taking a supernatant at 5 ℃ for later use, and preparing an R1 reagent;
2) firstly, 2-3g/L of bovine serum albumin, 2% of methylisothiazolinone in concentration and the balance of deionized water are added into pepsinogen I antibody emulsion solution, vortex oscillation is carried out for 20min, ultrasonic crushing is carried out for 10min, after the mixture is placed for 2h at 4 ℃, supernatant is taken and kept at 5 ℃ for later use, and an R2 reagent is prepared;
preparation of fatty alcohol and ethylene oxide condensate: the method comprises the steps of ring-opening polymerization of ethylene oxide in a reaction kettle, condensation with alcohol to generate fatty alcohol-polyoxyethylene ether, wherein the reaction kettle is in a vacuum state, the pressure value is 0.3MPa, the temperature is controlled to be 140 ℃, the time is 1 hour, and the heat preservation is continued for 2 hours after 1 hour.
Effect of different chyle concentrations on serum
The normal value range of the pepsinogen I is 70-200ug/L, the chyle content of a person is specially selected to be increased from 5 percent to 50 percent each time, the chyle content is compared with the common reagents in the market, the detection is repeatedly carried out at an interval of 60S in a continuous monitoring method, and the average value and the deviation are calculated.
Table 3 shows the results of the comparison of the reagent kit with the common detection agent on the market after the adding amount of chyle is increased by 5 percent
As shown in table 1, although the data detected at the two sides are not very different, which indicates that the stability of the two products is good in each detection, the detection effect of the comparison product is obviously different from that of the comparison product which is not added with chyle in the process of adding 5% -50% of chyle, so that the influence of the ratio of the plate product on the elimination of chyle in serum is obviously better than that of the comparison product.
In summary, the solution of the first embodiment is an optimal solution.
Taking the first example as an example, 10 groups of serum samples were extracted, wherein 5 groups of normal human serum and 5 groups of serum with gastritis were respectively detected separately from the product by common products on the market, the normal value was in the unit range of 70-200ug/L, gastritis ≧ 200ug/L was detected by liquid including serum and by continuous detection method, the interval time was 60S, as shown in table 4 below:
as shown in table 4 above, the product tends to be stable in the test of whether it has gastritis, better than the comparative product.
The above description is only for the specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto, and any changes or substitutions that are not thought of through the inventive work should be covered by the protection scope of the present invention.
Claims (7)
1. A pepsinogen I detection kit which is characterized in that: the reagent R1 comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises 50-150mmol/L of sodium phosphate buffer solution, 2-3g/L of bovine serum albumin, 2-3% of methylisothiazolinone, 1.0-5.0g/L of condensation product of fatty alcohol and ethylene oxide, 3-5mmol/L of magnesium chloride, 20-30mmol/L of ammonium formate and the balance of deionized water; the R2 reagent comprises 1.3-2mg/ml pepsinogen I antibody emulsion solution, 2-3g/L bovine serum albumin, 2-3% methylisothiazolinone in concentration and the balance of deionized water.
2. The pepsinogen I detection kit as claimed in claim 1, wherein: the pepsinogen I antibody emulsion solution comprises a 15% solution of latex particles coated by an anti-human pepsinogen I antibody, wherein the latex particles are monoclonal antibody cross-linked polymer nano microspheres which adopt polystyrene as a raw material, and the monoclonal antibody adopts a mouse or cattle antibody.
3. The pepsinogen I detection kit as claimed in claim 1, wherein: the preparation of the fatty alcohol and ethylene oxide condensation compound comprises the steps of ring-opening polymerization of ethylene oxide in a reaction kettle, condensation with alcohol to generate fatty alcohol-polyoxyethylene ether, wherein the reaction kettle is in a vacuum state, the pressure value is 0.3MPa, the temperature is controlled between 130-plus-material temperature and 150 ℃, the time is 1 hour, and the heat preservation is continued for 2 hours after 1 hour.
4. The pepsinogen I detection kit as claimed in claim 1, wherein: the temperature of the sodium phosphate buffer solution in the R1 reagent and the R2 reagent is 20 +/-1 ℃, and the pH is 8.
5. The pepsinogen I detection kit as claimed in claim 1, wherein: the sodium phosphate buffer solution is prepared by dissolving sodium phosphate in 1L of distilled water, then dropwise adding 1mol/L of KCl for preparation until the pH is 8, diluting with distilled water, storing in an ultraviolet sterilization cabinet for 30min, and storing in a 135 ℃ high-temperature sterilization box for 10 min.
6. The pepsinogen I detection kit as claimed in claim 1, wherein: the volume ratio of the R1 reagent to the R2 reagent is 2: 1.5.
7. The method for preparing a pepsinogen I detection kit as claimed in claim 1, which is characterized by comprising the following steps:
1) adding bovine serum albumin, a condensate of methyl isothiazolinone, fatty alcohol and ethylene oxide, magnesium chloride and ammonium formate into a sodium phosphate buffer solution, carrying out vortex oscillation for 10min, standing at 4-5 ℃ for 2h, centrifuging at 800R/min for 20min, standing for 2h, taking supernatant fluid for later use at 3-5 ℃ to prepare an R1 reagent;
2) firstly, 2-3g/L of bovine serum albumin, 2-3% of methylisothiazolinone and the balance of deionized water are added into pepsinogen I antibody emulsion solution, vortex oscillation is carried out for 20min, ultrasonic crushing is carried out for 10min, and after the mixture is placed for 2h at 4-5 ℃, supernatant is taken and is kept at 3-5 ℃ for later use to prepare R2 reagent;
3) and mixing the R1 reagent and the R2 reagent to obtain the pepsinogen I detection kit.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112255395A (en) * | 2020-12-23 | 2021-01-22 | 中生北控生物科技股份有限公司 | Method for eliminating chyle interference in lipemic sample, immunoturbidimetry kit and application |
| CN112255405A (en) * | 2020-10-27 | 2021-01-22 | 中拓医学检验有限公司 | Stable serum pepsinogen I determination kit, preparation method and application thereof |
-
2019
- 2019-12-15 CN CN201911287921.XA patent/CN111122867A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112255405A (en) * | 2020-10-27 | 2021-01-22 | 中拓医学检验有限公司 | Stable serum pepsinogen I determination kit, preparation method and application thereof |
| CN112255395A (en) * | 2020-12-23 | 2021-01-22 | 中生北控生物科技股份有限公司 | Method for eliminating chyle interference in lipemic sample, immunoturbidimetry kit and application |
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