CN112730833A - Ceruloplasmin determination kit by using immuno-transmission turbidimetry - Google Patents

Abstract

The invention provides a ceruloplasmin determination kit by an immune transmission turbidimetry method, belonging to the technical field of kits. The kit comprises an R1 reagent and an R2 reagent; the R1 reagent contains a first buffer solution, salt, a promoter, a first stabilizing agent and a first preservative; the R2 reagent contains a second buffer solution, salt, antiserum, a second stabilizing agent and a second preservative; the antiserum is selected from anti-human CER antiserum. The kit has the advantages of good stability and repeatability, accurate test result, simple preparation process of the reagent and contribution to production expansion. The experimental results show that: the reagent attenuation of the kit is less than 15 percent after 14 days of high temperature, the repeatability is less than or equal to 4.0 percent, and the kit can meet the clinical requirement.

Description

Ceruloplasmin determination kit by using immuno-transmission turbidimetry

Technical Field

The invention belongs to the technical field of kits, and particularly relates to a ceruloplasmin determination kit by an immune transmission turbidimetry method.

Background

Ceruloplasmin (CER), also known as copper oxidase (EC 1.16.3.1), is a copper-containing α 2 glycoprotein with a molecular weight of about 12-16 ten thousand, and is not easily purified. It is known that ceruloplasmin is a single-chain polypeptide containing 6-7 copper atoms per molecule, blue in color due to its copper content, contains approximately 10% sugars, and has a genetic polymorphism with terminal sialic acid attached to the polypeptide chain. The copper-containing enzyme protein has the functions of regulating the distribution of copper in various parts of an organism and synthesizing copper-containing enzyme protein, has the function of an antioxidant, has oxidase activity and has the capacity of catalyzing the oxidation of polyphenol and polyamine substrates. It is thought that ceruloplasmin is synthesized by the liver, and some ceruloplasmin is excreted in the biliary tract, and the content of ceruloplasmin in urine is very small. The determination of ceruloplasmin has certain significance for diagnosing certain diseases of liver, gallbladder, kidney and the like.

During the synthesis of ceruloplasmin by hepatocytes, copper infiltrates into the hepatocytes. After the ceruloplasmin is secreted from the liver, the ceruloplasmin is transported into tissues needing copper, and the copper is released in the process of metabolizing ceruloplasmin molecules. In addition to transporting copper, ceruloplasmin has a catalytic role in the oxidation of iron (Fe2+, Fe3+), polyamines, catecholamines and polyphenols.

In recent years, it has been considered that ceruloplasmin acts as an antioxidant. The antioxidant activity of ceruloplasmin in the blood circulation prevents the formation of lipid peroxides and free radicals in the tissues, which is of great importance especially in the case of inflammation. Ceruloplasmin is also an acute phase reaction protein. Ceruloplasmin is increased in the blood during infection, trauma and tumors. The most specific role is to assist in the diagnosis of Wilson's disease, i.e., a significant decrease in the amount of ceruloplasmin in the patient's blood, coupled with an increase in the amount of copper that can be dialyzed. Most patients may have impaired liver function with symptoms of the nervous system, if not timely treated, which is progressive and fatal, and therefore should be diagnosed in a timely manner and treated with the copper chelator, penicillamine. Ceruloplasmin is also often reduced in malnutrition, severe liver disease and nephrotic syndrome. The content of the contraceptive is obviously increased when the contraceptive is orally taken during the gestation period of women.

At present, the methods for detecting ceruloplasmin are mainly enzyme-linked immunosorbent assay and immunoturbidimetry, but the specific operation of the enzyme-linked immunosorbent assay is relatively complicated, the quantitative detection effect is poor, and the result is greatly influenced by human factors. The immunoturbidimetry method is simple to operate, can quickly and accurately detect the content of the ceruloplasmin within a few minutes, and is time-saving and labor-saving. The simplification of the immunoturbidimetry operation steps also correspondingly avoids the interference of a plurality of human operation factors and external factors such as reagents, environment and the like, has better stability and repeatability, and can reflect the content of the substance to be measured more truly. The method has low requirements on technology and equipment, can utilize a common biochemical analyzer for detection, is easy to realize automation, and can be popularized and applied in all levels of basic medical institutions.

Therefore, the development of a ceruloplasmin assay kit with simple preparation process, accurate result detection and stable performance is urgently needed.

Disclosure of Invention

The invention aims to provide a ceruloplasmin assay kit by an immune transmission turbidimetry method, which has the advantages of good stability and repeatability, simple preparation process and accurate result detection.

The invention provides a ceruloplasmin determination kit by an immune transmission turbidimetry, which comprises an R1 reagent and an R2 reagent;

the R1 reagent contains a first buffer solution, salt, a promoter, a first stabilizing agent and a first preservative;

the R2 reagent contains a second buffer solution, salt, antiserum, a second stabilizing agent and a second preservative;

the antiserum is selected from anti-human CER antiserum.

Preferably, the first buffer solution and the second buffer solution are selected from one or more of TRIS, BIS-TRIS, HEPES and TAPSO, and the concentration is 10-100 mM.

Preferably, the salt in the R1 reagent and the R2 reagent is NaCl, and the concentration is 0.5-1.0%.

Preferably, the accelerant is polyethylene glycol, and the concentration is 3% -10%.

Preferably, the first stabilizer and the second stabilizer are selected from aminocarboxylic acid chelating agents and queen flower series surfactants, and the concentration is 0.1-3%.

Preferably, the first preservative and the second preservative are Proclin300, and the concentration is 0.05-0.50%.

The invention has the advantages of

The invention provides a ceruloplasmin determination kit by an immune transmission turbidimetry, which comprises an R1 reagent and an R2 reagent; the R1 reagent contains a first buffer solution, salt, a promoter, a first stabilizing agent and a first preservative; the R2 reagent contains a second buffer solution, salt, antiserum, a second stabilizing agent and a second preservative; the antiserum is selected from anti-human CER antiserum. The kit has the advantages of good stability and repeatability, accurate test result, simple preparation process of the reagent and contribution to production expansion. The experimental results show that: the reagent attenuation of the kit is less than 15 percent after 14 days of high temperature, the repeatability is less than or equal to 4.0 percent, and the kit can meet the clinical requirement.

Drawings

FIG. 1 is a calibration graph of a ceruloplasmin assay kit according to example 1 of the present invention;

FIG. 2 is a calibration graph of the ceruloplasmin assay kit of example 2 of the present invention;

FIG. 3 is a graph showing the stability tracking of 13 months' worth of the ceruloplasmin assay kit of the present invention;

FIG. 4 shows the accelerated simulated attenuation change of the ceruloplasmin assay kit at 37 ℃ for 7 days and at 14 days;

FIG. 5 is a graph showing the stability tracking of the airborne 30-day assay value of the ceruloplasmin assay kit of the present invention at 2-8 ℃;

Detailed Description

The invention provides a ceruloplasmin determination kit by an immune transmission turbidimetry, which comprises an R1 reagent and an R2 reagent;

the R1 reagent contains a first buffer solution, salt, a promoter, a first stabilizing agent and a first preservative;

the R2 reagent contains a second buffer solution, salt, antiserum, a second stabilizing agent and a second preservative;

the antiserum is selected from anti-human CER antiserum, and the concentration is preferably 5-20%.

According to the invention, the first buffer solution and the second buffer solution can be the same or different, are preferably selected from one or more of TRIS, BIS-TRIS, HEPES and TAPSO, and have the concentration of preferably 10-100 mM.

According to the invention, the salt in the R1 reagent and the R2 reagent is preferably NaCl, and the concentration is preferably 0.5-1.0%.

According to the invention, the accelerator is preferably polyethylene glycol, preferably at a concentration of 3% to 10%.

According to the invention, the first stabilizer and the second stabilizer can be the same or different, are preferably selected from aminocarboxylic acid chelating agents and surfactants of queen series, and have a concentration of preferably 0.1-3%.

According to the invention, the first preservative and the second preservative may be the same or different, and are preferably Proclin300, preferably at a concentration of 0.05% to 0.50%.

The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified. The present invention is described in further detail below with reference to specific examples and with reference to the data. It will be understood that this example is intended to illustrate the invention and not to limit the scope of the invention in any way.

In the following examples, various procedures and methods not described in detail are conventional methods well known in the art. Materials, reagents, devices, instruments, apparatuses and the like used in the following examples are commercially available unless otherwise specified.

The invention is further illustrated by the following specific examples:

example 1: preparation of CER kit

R1 and R2 are buffers containing TRIS at pH 7.5 ± 0.05(25 ± 0.5 ℃).

Reagent R1:

TRIS	50mM
		NaCl	0.8％
polyethylene glycol	5％
		EDTA	0.5％
Proclin 300	0.1％
		pH	7.5±0.05(25±0.5℃)

Preparation of reagent R1: adding a full batch of purified water into a container, sequentially adding a full batch of TRIS, NaCl, EDTA, polyethylene glycol and Proclin300 into the container in sequence, stirring the mixture until the mixture is completely dissolved, adjusting the pH to 7.5 +/-0.05 (25 +/-0.5 ℃), stirring the mixture uniformly, re-measuring the pH to be in accordance with 7.5 +/-0.05 (25 +/-0.5 ℃), and filtering the mixture by using a 0.22 mu M filter membrane.

Reagent R2:

preparation of reagent R2: adding a full batch of purified water into a container, sequentially adding a full batch of TRIS, NaCl, Huawang and Proclin300 into the container, sequentially stirring until the materials are completely dissolved, adjusting the pH to 7.5 +/-0.05 (25 +/-0.5 ℃), uniformly stirring, repeatedly measuring the pH to be 7.5 +/-0.05 (25 +/-0.5 ℃), adding a full batch of CER antiserum into the container, slowly stirring until the materials are completely mixed, and filtering by using a 0.22 mu M filter membrane.

Example 2: preparation of CER kit

R1 and R2 are buffers containing TAPSO at pH 7.5 ± 0.05(25 ± 0.5 ℃).

Reagent R1:

TAPSO	30mM
		NaCl	0.6％
polyethylene glycol	5％
		EDTA	0.3％
Proclin 300	0.1％
		pH	7.5±0.05(25±0.5℃)

Preparation of reagent R1: adding a full batch of purified water into a container, sequentially adding a full batch of TAPSO, NaCl, EDTA, polyethylene glycol and Proclin300 into the container in sequence, stirring the mixture until the mixture is completely dissolved, adjusting the pH to 7.5 +/-0.05 (25 +/-0.5 ℃), stirring the mixture uniformly, measuring the pH to be in accordance with 7.5 +/-0.05 (25 +/-0.5 ℃), and filtering the mixture by using a 0.22 mu M filter membrane.

Reagent R2:

TAPSO	30mM
		NaCl	0.6％
antiserum	8％
		Flower king	0.5％
Proclin 300	0.1％
		pH	7.5±0.05(25±0.5℃)

Preparation of reagent R2: adding a full batch of purified water into a container, sequentially adding a full batch of TAPSO, NaCl, Huawang and Proclin300 into the container in sequence, sequentially stirring until the materials are completely dissolved, adjusting the pH to 7.5 +/-0.05 (25 +/-0.5 ℃), uniformly stirring, repeatedly measuring the pH to be 7.5 +/-0.05 (25 +/-0.5 ℃), adding a full batch of CER antiserum into the container, slowly stirring until the materials are completely mixed, and filtering by using a 0.22 mu M filter membrane.

Example 3 preparation of calibration Curve

Standard curves were prepared using the reagent R1 and the reagent R2 prepared in example 1, and Roche standards.

1. Opening the instrument to preheat for 15 minutes, adding items on the full-automatic biochemical analyzer, setting item parameters, registering reagent positions, placing R1 and R2 reagents, testing the residual quantity of the reagents, and ensuring that the reagents R1 and R2 are more than or equal to 15 mL/bottle.

2. Taking out the Roche calibrator, setting calibration parameters according to Roche instructions, detecting calibration samples by using a full-automatic biochemical analyzer at 0mg/L, 216.6mg/L, 451.25mg/L, 758.1mg/L and 1155.2mg/L, and making a standard curve by using a logarithmic linear equation for the absorbance change and the corresponding sample concentration, as shown in figure 1.

Standard curves were prepared for reagent R1 and reagent R2, prepared as in example 2, and Roche calibrators, as shown in FIG. 2.

Example 4 evaluation of reagent Properties

1. Evaluation of reproducibility

Repeated evaluations were carried out using the reagents prepared in example 1, and the measurements were repeated 10 times for 2 samples (130. + -.26 mg/L and 220. + -.44 mg/L) at different concentrations, and as shown in Table 1, the mean (M) and Standard Deviation (SD) of the results of the 10 measurements were calculated, and the coefficient of variation was obtained according to the following formula.

CV＝SD/M×100％

In the formula:

CV: coefficient of variation;

SD: standard deviation of 10 measurements;

m: average of 10 measurements.

TABLE 1 evaluation of reproducibility

As can be seen from the table, the repeatability CV of the detection result of the invention is less than 4.0 percent and meets the requirement.

2. Evaluation of shelf-Life stability

The stability of the reagent prepared in example 1 was evaluated for stability during expiration, the dispensed reagents were stored in an environment of 2 to 8 ℃ and were subjected to assay every 3 months for 13 months, and the change in the results of the reagents was observed to form a stability trace of the measured values, as shown in FIG. 3.

3. Evaluation of accelerated stability

The reagent R1 and the reagent R2 prepared in example 1 were placed in a 37 ℃ incubator (accelerated reagent) and a 4 ℃ refrigerator (refrigerated reagent), respectively, and the accelerated reagent and the refrigerated reagent were taken out on the 7 th day and the 14 th day, respectively, and the change in the results of the reagents was observed, as shown in FIG. 4.

4. Evaluation of on-board stability

The reagent R1 and the reagent R2 prepared in example 1 were decapped, placed in a fully automatic biochemical analyzer, and the decapped reagents and the non-decapped control reagents were taken out and tested simultaneously on the 7 th, 14 th, 21 st, 28 th, and 32 th days, respectively, and the change in the results of the reagents was observed, as shown in fig. 5.

In conclusion, the ceruloplasmin assay kit can be stored at 2-8 ℃ for at least 12 months without changing performance, can be stored at 37 ℃ for at least 14 days without changing performance, and can be stored on a vehicle for at least 30 days without changing performance. It is to be understood that the invention is not limited to the examples described above, but that modifications and variations are possible to those skilled in the art in light of the above teachings, and that all such modifications and variations are intended to be included within the scope of the invention as defined in the appended claims.

Claims (6)

1. A ceruloplasmin assay kit for immunoturbidimetry, comprising a reagent R1 and a reagent R2;

the R1 reagent contains a first buffer solution, salt, a promoter, a first stabilizing agent and a first preservative;

the R2 reagent contains a second buffer solution, salt, antiserum, a second stabilizing agent and a second preservative;

the antiserum is selected from anti-human CER antiserum.

2. The kit for assaying ceruloplasmin by using immunoturbidimetry according to claim 1, wherein the first buffer and the second buffer are selected from one or more of TRIS, BIS-TRIS, HEPES and TAPSO at a concentration of 10-100 mM.

3. The kit for assaying ceruloplasmin by immunoturbidimetry according to claim 1, wherein the salt of said R1 reagent and said R2 reagent is NaCl in a concentration of 0.5% to 1.0%.

4. The kit for assaying ceruloplasmin by using immunoturbidimetry according to claim 1, wherein said promoter is polyethylene glycol at a concentration of 3-10%.

5. The kit for assaying ceruloplasmin by using immunoturbidimetry according to claim 1, wherein said first and second stabilizers are selected from aminocarboxylic acid chelating agents and surfactants of the King series at a concentration of 0.1-3%.

6. The kit for assaying ceruloplasmin by using immunoturbidimetry according to claim 1, wherein said first and second preservatives are Proclin300 at a concentration of 0.05-0.50%.

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