CN112255404B

CN112255404B - Stable serum pepsinogen II determination kit, preparation method and application thereof

Info

Publication number: CN112255404B
Application number: CN202011161337.2A
Authority: CN
Inventors: 刘安娜
Original assignee: Zhongtuo Biotechnology Co ltd; Zhongtuo Medical Laboratory Co ltd
Current assignee: Zhongtuo Biotechnology Co ltd; Zhongtuo Medical Laboratory Co ltd
Priority date: 2020-10-27
Filing date: 2020-10-27
Publication date: 2021-05-18
Anticipated expiration: 2040-10-27
Also published as: CN112255404A

Abstract

The invention provides a pepsinogen II (PGII) determination kit, which comprises a reagent R1 and a reagent R2; the reagent R1 contains the following components: piperazine-1, 4-diethylsulfonic acid (PIPES) buffer solution, NaCl, sodium hydroxide, surfactant and preservative. Reagent R2: piperazine-1, 4-diethylsulfonic acid (PIPES) buffer solution, mouse anti-human PGII antibody coated latex particles, suspending agent, stabilizing agent and preservative. The invention also provides a preparation method and application of the kit, the kit can effectively avoid the interference of chylomicron, and different types of protective agents and suspending agents are adopted, so that the kit is a liquid kit with strong stability, high sensitivity, good repeatability and low cost.

Description

Stable serum pepsinogen II determination kit, preparation method and application thereof

Technical Field

The invention relates to the technical field of biochemical reagent determination, in particular to a pepsinogen II determination kit, and also relates to a preparation method and application of the pepsinogen II determination kit.

Background

Pepsinogen (pepsinogen), synthesized by the main cells of the secretory glands, is converted into pepsin in the stomach cavity by the action of hydrochloric acid (HCL) or already active pepsin (pepsin), decomposing the protein into fat, peptone and a small amount of polypeptides. The optimum pH for the action of this enzyme is 2, and after entering the small intestine, the enzyme activity is lost.

Pepsinogen is a precursor of pepsin, which is divided into 2 subgroups according to its biochemical properties and immunogenicity, and 1-5 components have the same immunogenicity, are called pepsinogen I and are mainly secreted by the main cells of the fundus stomach gland and the mucous neck cells;

components

6 and 7 are called pepsinogen II, which is produced in addition to secretion by the principal cells of the fundic gland and the mucous cervical cells of the pyloric glands of the cardia and antrum and in the upper duodenum.

The serum pepsinogen levels reflect the morphology 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; progressive reduction of the PGI/II ratio correlates with progression of gastric mucosal atrophy. Thus, a combined determination of the ratio of PGI and PGII may serve as a "serological biopsy" of the mucosa of the gastric fundus gland. The serum measured values of the composition are different, and the composition has different changes in various stomach diseases, thereby providing reliable diagnostic value for clinic.

The existing methods for detecting the pepsinogen II include a latex immunoturbidimetry method, a double antibody sandwich Immunochemiluminometry (ILMA) method and a colloidal gold colorimetric method. The double antibody sandwich Immunochemiluminescence (ILMA) method has high accuracy and sensitivity, but the instrument and equipment are expensive, the reagent is inconvenient to store, and the price is high; the colloidal gold colorimetric method is simple to operate, has a fast result, but can only be used for qualitative research and cannot be used for quantitative analysis. The latex immunoturbidimetry method has the advantages of high specificity, simple and rapid operation, accuracy and safety, capability of automatic analysis and lower cost, but the kit produced in the prior art has the defects of low analysis sensitivity, poor stability, easy influence by Chylomicron (CM) and the like.

Disclosure of Invention

In order to solve the problems, the invention provides a pepsinogen II determination kit, a preparation method and application thereof.

The invention is realized by the following technical scheme:

a pepsinogen II determination kit comprises a reagent R1 and a reagent R2;

the reagent R1 contains the following components:

buffer solution is 20-50 mmol/L;

NaCl 1-9g/L；

sodium hydroxide0.1g /L；

0.1 to 2 percent of surfactant;

0.5-1g/L of preservative;

the reagent R2 contains the following components:

buffer solution is 20-50 mmol/L;

20-50mg/L of latex particles coated by a mouse anti-human PGII antibody;

10-20g/L of stabilizer;

0.1 to 2 percent of surfactant;

10-20g/L of suspending agent;

0.5-1g/L of preservative;

wherein the percentage of surfactant is by volume.

Preferably, the pH of the reagent R1 is 6.5-7.5.

Preferably, the pH of the reagent R2 is 6.8-7.5.

Preferably, the surfactant in the reagents R1 and R2 is selected from one or more of polyoxyethylene sorbitol hexastearate, triton x-100 and propylene glycol fatty acid ester. More preferably, the surfactant in the reagent R1 is polyoxyethylene sorbitol hexastearate and propylene glycol fatty acid ester, and the surfactant in the reagent R2 is triton 100.

Preferably, the stabilizer in the reagent R2 is one or more of bovine serum albumin, polyvinylpyrrolidone k30 and polyethylene glycol 4000. More preferably, the stabilizer consists of bovine serum albumin and polyvinylpyrrolidone k 30.

Preferably, the suspending agent in the reagent R2 is one or more of trehalose, mannitol and glycerol. More preferably, the suspending agent is compounded by mannitol and glycerol.

Preferably, the latex particle diameter in the reagent R2 is one or more of 50nm, 100nm and 150 nm. More preferably, the latex particles are 100nm in diameter.

Preferably, the preservative in the reagents R1 and R2 is one or more of sodium azide, proclin300, MIT, and sodium benzoate, more preferably, the preservative is proclin 300.

Preferably, the volume ratio of the reagent R1 to the reagent R2 is 1-5: 1. More preferably, the volume ratio of reagent R1 to reagent R2 is 5: 1.

The preparation method of the pepsinogen II determination kit comprises the following steps: the preparation method of the mouse anti-human pepsinogen II antibody coated latex particle comprises the following steps: taking a proper amount of polystyrene latex particles with carboxylated surfaces, adding the polystyrene latex particles into 10ml of buffer solution to ensure that the final concentration of the latex particles is 1.0%; then adding a proper amount of the mouse anti-human pepsinogen II antibody, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), mixing and stirring for about 3 hours at room temperature, adding 1ml of 10g/L BSA solution, centrifuging for 40 minutes at 12000 rpm, removing the supernatant, and obtaining the precipitate, namely the mouse anti-human pepsinogen II antibody coated latex particles. And adding other substances according to the proportion to dissolve the pepsinogen II to prepare the pepsinogen II determination kit.

The invention also discloses the application of the pepsinogen II determination kit, which is used for determining the concentration of pepsinogen II in serum for the purposes of diagnosis and treatment of non-diseases.

The kit adopts a latex immunoturbidimetry method, and has the reaction principle that PGII in a sample can generate agglutination reaction of antigen antibodies with anti-PGII antibodies adsorbed on latex particles to form an immune complex, and the content of PGII in the sample can be calculated by measuring the change of absorbance of the immune complex. The reaction has specificity, the full-automatic biochemical analyzer is used for detection, the operation is simple, the automation degree is high, the human error can be reduced, and the method is suitable for most clinical laboratories.

Advantageous effects

1) The stable pepsinogen II determination kit is a liquid double reagent, does not need to be re-dissolved for preparation, and can be directly used after opening a bottle.

2) The bovine serum albumin and the polyvinylpyrrolidone k30 are added into the reagent R2 to form a composite stabilizer, the mannitol and the glycerol form a composite suspending aid, and the components have synergistic effect, so that the stability of the antibody latex particles in the reagent is effectively improved, the stability of the reagent is excellent, and the reagent is further promoted in the market.

3) The polyoxyethylene sorbitol hexastearate and the propylene glycol fatty acid ester are added to act together, so that chylomicron can be better emulsified, the chylomicron can be effectively shielded, interference is avoided, the anti-interference capability of the reagent is greatly enhanced, and the stability of the reagent is improved to a certain extent.

4) The reagent has excellent performance indexes such as accuracy, repeatability, analysis sensitivity, linear range, stability and the like, is low in price and convenient to use, and is favorable for further popularization of the reagent in the market.

Drawings

FIG. 1 is a correlation curve for the reagents of example 1 and comparative example 1;

FIG. 2 is a linear curve of example 1;

FIG. 3 is a graph showing the change in concentration of the pepsinogen II measuring reagent of example 1 and comparative examples 1, 2, 3 and 4 for stability test.

Detailed Description

The invention is further illustrated by the following specific examples:

in the use of the kit of this embodiment, the determination method is to use a michael 800 full-automatic biochemical analyzer with double reagent functions, and perform determination by using a rate method, where the dominant wavelength is 700nm, and the operations are as follows:

adding 6 muL of physiological saline, a sample or a calibrator, adding 150 muL of R1 reagent, pre-incubating for 5min, adding 30 muL of R2 reagent, mixing uniformly, delaying for 1min, reading absorbance A1, reading absorbance A2 after 5min,

calculating Δ A = (A2-A1).

Pepsinogen II content (ng/mL) = (Δ a samples ÷ Δ a calibrant) × calibrant concentration.

Sample requirements:

1. insoluble blood serum.

2. Sample stability: the specimen can be stored stably for 3 days at the temperature of 2-8 ℃ and for 2 weeks at the temperature of-20 ℃.

Example 1

A conventional pepsinogen II assay kit comprises a reagent R1 and a reagent R2.

The reagent R1 contains the following components:

piperazine-1, 4-diethylsulfonic acid (PIPES) buffer 50 mmol/L;

NaCl 9g/L；

sodium hydroxide0.1g /L；

1% of propylene glycol fatty acid ester;

1% of polyoxyethylene sorbitol hexastearate;

proclin300 1 ml/L。

the pH of reagent R1 was 7.2.

The reagent R2 contains the following components:

piperazine-1, 4-diethylsulfonic acid (PIPES) buffer 50 mmol/L;

30mg/L of mouse anti-human PGII antibody coated latex particles;

bovine Serum Albumin (BSA) 5 g/L;

polyvinylpyrrolidone k 305 g/L;

5g/L of mannitol;

glycerol is 10 ml/L;

triton 1001%;

proclin300 1ml/L。

the pH of reagent R2 was 6.8.

Wherein the percentages are by volume.

Comparative example 1

Commercially available imported Sigma pepsinogen II assay kit.

Comparative example 2

The difference from the pepsinogen II assay kit in example 1 is only that the suspending agent in reagent R2 is mannitol in its entirety. The rest is the same as in example 1.

Comparative example 3

The kit is different from the kit for assaying pepsinogen II in example 1 only in that the suspending agent in the reagent R2 is glycerol, and the other steps are the same as those in example 1.

Comparative example 4

The difference from the pepsinogen II assay kit of example 1 is that there is no suspending agent in the reagent R2, and the other steps are the same as in example 1.

Comparative example 5

The kit is different from the pepsinogen II determination kit in the example 1 only in that the reagent R1 does not contain polyoxyethylene sorbitol hexastearate, and the other steps are the same as the example 1.

Comparative example 6

The kit is the same as that used in example 1 except that the reagent R1 does not contain a propylene glycol fatty acid ester.

Comparative example 7

The kit is different from the kit for assaying pepsinogen II in example 1 only in that the reagent R1 does not contain polyoxyethylene sorbitol hexastearate or propylene glycol fatty acid ester, and the other steps are the same as those in example 1.

Performance verification

Test No.)

Correlation experiments: example 1 and comparative example 1, 40 clinical serum samples were tested simultaneously, correlation analysis was performed on the two sets of test results, and a correlation coefficient r was calculated; the relative deviation (r) of 40 pairs of data was calculated using the test results of comparative example 1 as control values, respectively. It is required that r is not less than 0.990 and the relative deviation is not more than. + -. 10%.

The results are shown in table 1, and correlation curves (shown in fig. 1) were obtained for the reagents of example 1 and comparative example 1.

TABLE 1 correlation comparative experiment results

As can be seen from Table 1 and FIG. 1, the maximum value of the deviation of the test addresses of the serum of the kits of example 1 and comparative example 1 is-7.69%, the correlation between high values is good, the correlation coefficient of the two reagents is 0.998, and the test results of example 1 and comparative example 1 are very close to each other, so that the test reagent of example 1 provided by the invention has good correlation with the imported test reagent, and can completely replace the imported reagent for clinical examination.

Test No. two

And (3) precision test: in example 1 and comparative example 1, 20 tests were performed on clinical samples, and the mean, standard deviation, and coefficient of variation of the 20 tests were calculated. The results are shown in Table 2.

TABLE 2 precision testing data table

As can be seen from Table 2, the standard deviation of the detection values of example 1 and comparative example 1 is small, the coefficient of variation is small, the repeatability is good, and the precision is high. Example 1 precision is a complete replacement for imported reagents.

Experiment three

Linear experiments: taking 80ng/mL of the pepsinogen II high-value sample, diluting, preparing 6 samples with different concentrations, sequentially taking samples with the concentrations of 80ng/mL, 40ng/mL, 20ng/mL, 10ng/mL, 5ng/mL and 0ng/mL, detecting by using the reagent of the embodiment 1, respectively measuring each sample at each concentration level for three times, and respectively taking the average value. The results are shown in Table 3.

TABLE 3 table of testing data of linear correlation verification experiment

As can be seen from Table 3 and FIG. 2, the linear variation of example 1 of the present invention with diluted concentration is achieved, the linear correlation coefficient is 0.998 and greater than 0.990, the standard requirement is met, and the value is greater than that of other control groups. The linear range is better in example 1. The better linear variation of the reagent of example 1 is more consistent with the requirements of clinical case samples.

Experiment four

Stability test: the pepsinogen II assay reagents provided in example 1 and comparative examples 1, 2, 3, 4 were subjected to a stability test according to the following protocol: the reagents provided in example 1 and comparative examples 1, 2, 3 and 4, each of which comprises 10 groups in parallel, were stored together in a 37 ℃ incubator, subjected to an accelerated test, and a quality control with a target value of 20. + -. 2.0mg/L was tested 5 times per day, and each group was averaged, and the change in the measured value of the quality control was monitored, and the results are shown in Table 4.

Table 4 reagent thermal stability verification data

As can be seen from Table 4 and FIG. 3, the reagent of example 1 provided by the present invention has substantially no change within 10 days at 37 ℃, and has good stability; while the agent of comparative example 4 changed significantly within 10 days, comparative examples 2, 3 with a single suspending agent were superior to comparative example 4 without suspending agent. The stability of the kit of example 1 is superior to that of the kits of comparative examples 1, 2, 3 and 4, which shows that the stability of the pepsinogen II determination kit is obviously improved by the combined action of the buffer solution, various protective agents and the suspending agent.

Experiment five

Interference experiments: and (3) adding the traceability quality control substances and the median quality control substances (the target value is 20 +/-2.0 ng/mL) into the content of the chylomicron in the table respectively. Then, the content of pepsinogen II in the sample was measured by using the reagent of example 1, the reagent of comparative example 5, the reagent of comparative example 6 and the reagent of comparative example 7, and the measurement results of each group are shown in Table 5.

TABLE 5 anti-interference verification results of reagents

As can be seen from Table 5, when the chylomicron is 0.5mL/L, the reagent of example 1 is not significantly disturbed, the reagents of comparative examples 1, 5 and 6 are somewhat disturbed, and the disturbance of comparative example 7 is more significant. When the chylomicron is 2mL/L, the reagent of the example 1 is not obviously interfered, and the reagents of the comparative examples 1, 5, 6 and 7 are obviously interfered. Example 1 has a stronger interference resistance than comparative examples 5, 6 and 7, and example 1 having two components has a stronger interference resistance. The result shows that the anti-interference performance of the reagent in the embodiment 1 is obviously improved by the synergistic effect of the polyoxyethylene sorbitol hexastearate and the propylene glycol fatty acid ester after the polyoxyethylene sorbitol hexastearate and the propylene glycol fatty acid ester are added, and the reagent is superior to the reagent in the comparative example 1 and meets the clinical requirements.

In conclusion, the kit adopts polyoxyethylene sorbitol hexastearate, propylene glycol fatty acid ester, different protective agents and suspending agents, and is a liquid kit with high anti-interference performance, high stability, high sensitivity, good repeatability and low cost. Provides good development space for the kit and simultaneously enhances the market competitiveness of the kit.

The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.

While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

Claims

1. A pepsinogen II determination kit is characterized in that the kit comprises a reagent R1 and a reagent R2;

the reagent R1 contains the following components:

piperazine-1, 4-diethylsulfonic acid (PIPES) buffer 50 mmol/L;

NaCl 9g/L；

0.1 g/L of sodium hydroxide;

1% of propylene glycol fatty acid ester;

1% of polyoxyethylene sorbitol hexastearate;

proclin300 1 ml/L；

the reagent R2 contains the following components:

piperazine-1, 4-diethylsulfonic acid (PIPES) buffer 50 mmol/L;

30mg/L of mouse anti-human PGII antibody coated latex particles;

bovine Serum Albumin (BSA) 5 g/L;

polyvinylpyrrolidone k 305 g/L;

5g/L of mannitol;

glycerol is 10 ml/L;

triton 1001%;

proclin300 1ml/L；

the volume ratio of the reagent R1 to the reagent R2 is 5:1, the pH value of the reagent R1 is 7.2, and the pH value of the reagent R2 is 6.8, wherein the percentage is the volume ratio.

2. A method for preparing the pepsinogen II assay kit as defined in claim 1, which comprises: the method comprises the following steps: firstly preparing a mouse anti-human pepsinogen II antibody coated latex particle: taking a proper amount of polystyrene latex particles with carboxylated surfaces, adding the polystyrene latex particles into 10ml of buffer solution to ensure that the final concentration of the latex particles is 1.0%; then adding a proper amount of mouse anti-human pepsinogen II antibody, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, mixing and stirring for 3 hours at room temperature, adding 1ml of 10g/L BSA solution, centrifuging for 40 minutes at 12000 rpm, removing supernatant, and obtaining precipitate, namely mouse anti-human pepsinogen II antibody coated latex particles; adding other substances according to the proportion to dissolve, and preparing a pepsinogen II determination kit; the latex particle diameter in the reagent R2 is one or more of 50nm, 100nm and 150 nm.

3. Use of the pepsinogen II assay kit of claim 1 for determining the pepsinogen II concentration in serum for non-disease diagnostic and therapeutic purposes.