阅读说明：本技术 一种铜蓝蛋白检测试剂盒及其制备方法 (Ceruloplasmin detection kit and preparation method thereof ) 是由 李媛媛 于 2021-09-18 设计创作，主要内容包括：本发明涉及医学检测技术领域,尤其涉及一种铜蓝蛋白检测试剂盒。该试剂盒由R1试剂和R2试剂组成；所述试剂R1由pH7.0～8.0的5～30mMPB缓冲液、9g/L-30g/L的无机盐、40-80g/L促凝剂、0.5～2g/L表面活性剂和0.1-1g/L的防腐剂组成；所述R2试剂由pH7.0～8.0的5-100mM缓冲液、9-30g/L的无机盐、100～500g/L铜蓝蛋白多抗血清、0-5g/L保护剂、60～80g/L稳定剂和0.1-1g/L防腐剂组成；所述R2中稳定剂为蔗糖、海藻糖、甘油、葡萄糖的一种或几种。实验表明,本发明所述试剂盒稳定性好、批间差异小、灵敏度高,有利于临床上广泛推广和使用。(The invention relates to the technical field of medical detection, in particular to a ceruloplasmin detection kit. The kit consists of an R1 reagent and an R2 reagent; the reagent R1 consists of 5-30 mMPB buffer solution with the pH value of 7.0-8.0, 9-30g/L inorganic salt, 40-80g/L coagulant, 0.5-2 g/L surfactant and 0.1-1g/L preservative; the R2 reagent consists of 5-100mM buffer solution with the pH value of 7.0-8.0, 9-30g/L inorganic salt, 100-500 g/L ceruloplasmin multiple antiserum, 0-5g/L protective agent, 60-80 g/L stabilizer and 0.1-1g/L preservative; the stabilizer in the R2 is one or more of sucrose, trehalose, glycerol and glucose. Experiments show that the kit has good stability, small batch difference and high sensitivity, and is favorable for wide clinical popularization and use.)

1. A detection kit for ceruloplasmin is characterized by consisting of an R1 reagent and an R2 reagent;

the reagent R1 consists of 5-30mM PB buffer solution with the pH value of 7.0-8.0, 9-30g/L inorganic salt, 40-80g/L coagulant, 0.5-2 g/L surfactant and 0.1-1g/L preservative;

the R2 reagent consists of 5-100mM buffer solution with the pH value of 7.0-8.0, 9-30g/L inorganic salt, 100-500 g/L ceruloplasmin multiple antiserum, 0-5g/L protective agent, 60-80 g/L stabilizer and 0.1-1g/L preservative;

the stabilizer in the R2 is one or more of sucrose, trehalose, glycerol and glucose.

2. The test kit of claim 1, wherein the preservative in the R1 reagent and the R2 reagent is ProClin 300.

3. The detection kit according to claim 1, wherein the inorganic salt in the R1 reagent and the R2 reagent is NaCl and/or KCl.

4. The test kit according to claim 1, wherein in the R1 reagent:

the coagulant is polyethylene glycol 6000 and/or dextran;

the surfactant is Tween 20 and/or Tween 80.

5. The detection kit according to claim 1, wherein, in the R2 reagent,

the buffer solution is one or more of acetate buffer solution, ammonium chloride buffer solution, phosphate buffer solution, TRIS buffer solution, boric acid buffer solution, glycine buffer solution, CAPSO, MOPS or Hepes buffer solution;

the inorganic salt is NaCl and/or KCl;

the protective agent is one or more of BSA, casein and casein.

6. The detection kit according to claim 1, wherein the ceruloplasmin is goat, rabbit, horse, mouse or other animal anti-human multi-antisera.

7. The kit according to any one of claims 1 to 6, which consists of an R1 reagent and an R2 reagent;

the reagent R1 consists of a 5-30mM PB buffer solution with the pH value of 7.0-8.0, 9-30g/L sodium chloride, 40-80g/L polyethylene glycol, 0.5-2 g/L Tween 20 and 0.1-1g/L ProClin 300;

the R2 reagent consists of 5-100mM TRIS buffer solution or MOPS buffer solution with the pH value of 7.0-8.0, 9-30g/L sodium chloride, 100-500 g/L ceruloplasmin polyclonal antiserum, 0-5g/LBSA, 60-80 g/L sucrose and 0.1-1g/L ProClin 300.

8. The kit of claim 7, consisting of an R1 reagent and an R2 reagent;

the reagent R1 consists of a 5-30mM PB buffer solution with the pH value of 7.4, 9-20 g/L sodium chloride, 60-70g/L polyethylene glycol, 0.5-1.5g/L Tween 20 and 0.5-1g/L ProClin 300;

the R2 reagent consists of 10-100mM TRIS buffer solution or MOPS buffer solution with the pH value of 7.4, 9g/L sodium chloride, 150g/L ceruloplasmin polyclonal antiserum, 0-5g/L BSA, 60-80 g/L sucrose and 0.1-1g/L ProClin 300.

Technical Field

The invention relates to the technical field of medical detection, in particular to a ceruloplasmin detection kit and a preparation method thereof.

Background

Ceruloplasmin (CER), also known as hemocyanin and copper oxidase, is a copper-containing alpha 2-globulin with oxidase and has a relative molecular mass of 151 × 10³Each molecule contains 6-7 copper atoms, the copper content is 0.34%, and about 95% of copper in serum and alpha 2-globulin are combined into ceruloplasmin to play the role of regulating iron absorption and transportation, and the ceruloplasmin has the activities of ferrous oxidase and amine chloride. Its main physiological action includes transferring copper and iron oxide, promoting cell growth and angiogenesis, etc., and copper is one of the trace elements essential for human body, possesses important biological function and has the mutual regulation action with thyroxine. It can promote decomposition of histamine, serotonin, etc., and can form nonspecific disease resistance of organism. It is generally accepted that ceruloplasmin is synthesized by the liver, aSome of the urine is excreted from the biliary tract, and the urine content is very small. The determination of ceruloplasmin has certain significance for diagnosing certain diseases of liver, gallbladder, kidney and the like.

At present, the common method for detecting ceruloplasmin is a common immunoturbidimetry, which can not only meet the clinical requirements on the sensitivity of reagents, but also has simple preparation operation of the reagents and accurate and rapid detection of results. However, the multi-antiserum, which is a key raw material used in the common immunoturbidimetry method, is different due to uncontrollable factors, so that the performance of the reagent is influenced. And ceruloplasmin polyclonal antisera are not stable in buffer for long periods of time (18 months).

Disclosure of Invention

In view of the above, the invention provides a ceruloplasmin detection kit and a preparation method thereof, and the kit has small batch difference, high sensitivity and good stability, and can be widely used in clinical application.

The invention provides a detection kit for ceruloplasmin, which consists of an R1 reagent and an R2 reagent;

the reagent R1 consists of 5-30mM PB buffer solution with the pH value of 7.0-8.0, 9-30g/L inorganic salt, 40-80g/L coagulant, 0.5-2 g/L surfactant and 0.1-1g/L preservative;

the R2 reagent consists of 5-100mM buffer solution with the pH value of 7.0-8.0, 9-30g/L inorganic salt, 100-500 g/L ceruloplasmin multiple antiserum, 0-5g/L protective agent, 60-80 g/L stabilizer and 0.1-1g/L preservative;

the stabilizer in the R2 is one or more of sucrose, trehalose, glycerol and glucose.

The kit of the invention is used for determining ceruloplasmin based on an immunoturbidimetry, and the detection principle is as follows:

and (3) combining the reagent R2 with the ceruloplasmin in the sample to perform agglutination reaction to form an antigen-antibody immune complex and generate turbidity, wherein the turbidity is related to the concentration of the ceruloplasmin in the sample, the absorbance value at the moment is measured, and the content of the ceruloplasmin is calculated according to the calibration curve.

Specifically, the use method of the kit comprises the following steps:

firstly, uniformly mixing a reagent R1 and a CER serum sample to be detected for 5 minutes, and then adding a reagent R2 to start reaction, wherein the reagent R1, the reagent R2 and the sample are 250:50:2 in volume ratio; and (3) determining the initial absorbance A1 by comparing with a blank at 5 minutes, then determining the absorbance A2 by comparing with the blank at 10 minutes, calculating the difference delta A between A2 and A1, and obtaining the CER content in the CER serum sample to be detected according to the standard curve.

In some embodiments, the preservative in the R1 reagent and the R2 reagent is ProClin 300.

In some embodiments, the inorganic salt in the R1 reagent and the R2 reagent is NaCl and/or KCl.

In some embodiments, the R1 reagent wherein:

the coagulant is polyethylene glycol 6000 and/or dextran;

the surfactant is Tween 20 and/or Tween 80.

In the invention, the buffer solution of the R1 reagent can effectively solve the problem of reagent batch-to-batch difference. In some embodiments, the buffer for the R1 reagent is preferably 10mM PB buffer, pH 7.4.

In some embodiments, in the R2 reagent,

the buffer solution is one or more of acetate buffer solution, ammonium chloride buffer solution, phosphate buffer solution, TRIS buffer solution, boric acid buffer solution, glycine buffer solution, CAPSO, MOPS or Hepes buffer solution;

the inorganic salt is NaCl and/or KCl;

the protective agent is one or more of BSA, casein and casein.

In some embodiments, the ceruloplasmin polyclonal antiserum is a sheep anti-human, rabbit anti-human, horse anti-human, mouse anti-human or other animal anti-human polyclonal antiserum. Before use, the ceruloplasmin multiple antiserum is subjected to centrifugal treatment and filtration, so that the inter-batch difference of the reagent is reduced, and the stability of the reagent is improved.

In the invention, the inorganic salt and the stabilizing agent are matched with other components according to a specific proportion, so that the stability and the batch difference of the reagent are obviously improved.

Specifically, the kit of the present invention consists of an R1 reagent and an R2 reagent;

the reagent R1 consists of a 5-30mM PB buffer solution with the pH value of 7.0-8.0, 9-30g/L sodium chloride, 40-80g/L polyethylene glycol, 0.5-2 g/L Tween 20 and 0.1-1g/L ProClin 300;

the R2 reagent consists of 5-100mM TRIS buffer solution or MOPS buffer solution with the pH value of 7.0-8.0, 9-30g/L sodium chloride, 100-500 g/L ceruloplasmin polyclonal antiserum, 0-5g/LBSA, 60-80 g/L sucrose and 0.1-1g/L ProClin 300.

In some embodiments, the kit consists of R1 reagent and R2 reagent;

the reagent R1 consists of a 5-30mM PB buffer solution with the pH value of 7.4, 9-20 g/L sodium chloride, 60-70g/L polyethylene glycol, 0.5-1.5g/L Tween 20 and 0.5-1g/L ProClin 300;

the R2 reagent consists of 10-100mM TRIS buffer solution or MOPS buffer solution with the pH value of 7.4, 9g/L sodium chloride, 150g/L ceruloplasmin polyclonal antiserum, 0-5g/L BSA, 60-80 g/L sucrose and 0.1-1g/L ProClin 300.

Compared with the prior art, the invention has the following beneficial effects:

(1) the kit prepared by the method has detection sensitivity and accuracy meeting clinical requirements.

(2) The formula provided by the invention can eliminate the batch-to-batch difference of different batches of the multiple antiserum on the performance of the kit.

(3) The kit has excellent stability. The invention adopts an immunoturbidimetry method, and finds out a better proportion to improve the stability of the reagent by optimizing a reaction system and adopting a plurality of stabilizers as a reagent R2; the ceruloplasmin multiple antiserum adopts a better treatment mode, and obviously improves the stability and the batch difference of the reagent.

(4) The kit prepared by the method can be used on a full-automatic biochemical analyzer, is suitable for full-automatic testing, and can be developed and popularized on a large scale.

The invention adopts the innovation points as follows:

1) the present invention effectively eliminates reagent lot-to-lot variation using 10mM PB pH7.0-8.0 as the R1 buffer.

2) The stabilizer with the optimal proportion is added into the reagent R2 of the kit, and the stability of the ceruloplasmin multiple antiserum is enhanced and the storage life is prolonged by adopting an optimal treatment mode.

Drawings

FIG. 1 shows a scatter plot of CER concentration versus measured Δ A values;

FIG. 2 shows the correlation analysis results of the kit of example 1 of the present invention and the control CER kit (Normal immunoturbidimetry);

FIG. 3 shows the correlation analysis results of the kit of example 2 of the present invention and the control CER kit (Normal immunoturbidimetry);

FIG. 4 shows the correlation analysis results of the kit of example 3 of the present invention and the control CER kit (Normal immunoturbidimetry);

FIG. 5 shows the results of correlation analysis of different batches of a control CER kit;

FIG. 6 shows the results of the correlation analysis of the kits of example 1 and example 2;

FIG. 7 shows the results of the correlation analysis of the kits of example 1 and example 3;

FIG. 8 shows the results of the correlation analysis of the kits of example 2 and example 3.

Detailed Description

The invention provides a ceruloplasmin detection kit. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. 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 test materials adopted by the invention are all common commercial products and can be purchased in the market.

The invention is further illustrated by the following examples:

EXAMPLE 1 preparation of the kit of the invention

The kit is used for determining CER based on a common immunoturbidimetry, and consists of a reagent R1 and a reagent R2, wherein the buffer solution in the reagent R1 is 10mM PB buffer solution with pH 7.4. Reagent R2 is sucrose. However, 3 different batches of ceruloplasmin polyclonal antiserum (Origene, lot # F00702-01; C14503R-01; C14503-01) were used as the components of reagent R2, and were labeled as batch 1, batch 2, and batch 3.

TABLE 1 composition of reagent R1

The reagent R1 was prepared by the following steps (preparation 1L): respectively preparing a PB buffer solution by using purified water according to the component concentrations of examples 1-3 in the table 1 and the component content of the reagent R1, and adjusting the pH to be used as an R1 buffer solution; then adding corresponding amount of sodium chloride, tween 20, polyethylene glycol 6000 and ProClin300, mixing uniformly and adjusting pH with sodium hydroxide/hydrochloric acid to obtain a reagent R1.

The composition of the reagent R2 is shown in table 2.

TABLE 2 composition of reagent R2

The reagent R2 was prepared by the following steps (preparation 1L): preparing a buffer solution by using purified water according to the component content of the reagent R2, and adjusting the pH value to obtain a R2 preservation solution; dissolving the processed ceruloplasmin polyclonal antiserum into R2 preservation solution according to the component content of the reagent R2, and uniformly stirring to obtain a R2 reagent;

comparative examples 1-2 kit and preparation

The kit can be used for measuring CER by a common immunoturbidimetry method and comprises a reagent R1 and a reagent R2.

TABLE 3 composition of reagent R1

The reagent R1 was prepared by the following steps (preparation 1L): preparing PB buffer solution by using purified water according to the component concentrations of comparative examples 1-2 in the table 1 and the component content of the reagent R1, and adjusting the pH to be used as R1 buffer solution; then adding corresponding amount of sodium chloride, tween 20, polyethylene glycol 6000 and ProClin300, mixing uniformly and adjusting pH with sodium hydroxide/hydrochloric acid to obtain a reagent R1.

The composition of the reagent R2 is shown in Table 4

TABLE 4 composition of reagent R2

The reagent R2 was prepared by the following steps (preparation 1L): preparing a buffer solution by using purified water according to the component content of the reagent R2, and adjusting the pH value to obtain a R2 preservation solution; and (3) dissolving the processed ceruloplasmin polyclonal antiserum into R2 preservation solution according to the component content of the reagent R2, and uniformly stirring to obtain the R2 reagent.

Effect test example 1

Effect test kit: the kits and control CER kits (ordinary immunoturbidimetry) of examples 1 to 3, respectively, Ilikang, Zhejiang, Biotechnology Ltd.

The instrument comprises the following steps: toshiba 120 biochemical analyzer.

And (3) testing wavelength: the main wavelength is 340nm, and the sub-wavelength is 700 nm.

The test method comprises the following steps: end point method, incremental reaction. At 37 ℃, firstly, uniformly mixing a reagent R1 and a serum sample of CER for 5 minutes, and then adding a reagent R2 to start a reaction, wherein the volume ratio of the reagent R1 to the reagent R2 to the sample is 250:50: 2; the initial absorbance A1 was measured against the blank at 5 minutes, then the absorbance A2 was measured against the blank at 10 minutes, and the difference Δ A between A2 and A1 was calculated.

1. Determination of the Standard Curve

The standard serum samples of a series of standard concentrations of CER shown in Table 5 were individually measured for delta A values using the CER kits of examples 1-3 and the control CER, and a standard curve for each kit was fitted based on the series of CER concentrations and the measured delta A values, respectively, with the standard curves of examples 1-3 being SPLINE functions.

Fig. 1 shows a scattergram of the correlation between the CER concentration and the measured Δ a value, and it can be seen from fig. 1 that in examples 1 to 3, the absorbance change is more significant and the sensitivity is higher than that of the control under the same conditions.

TABLE 5 results of the measurement of the Delta A values of the Standard serum samples

As can be seen from FIG. 1, the formulation provided by the present invention can ensure that the differences of the multiple antiserum raw materials of different batches do not affect the calibration result of the kit. Sucrose and BSA mainly act to protect the polyclonal antisera, i.e. to improve reagent stability.

2. A sample with ceruloplasmin concentration of 40mg/dL was prepared, and the sample was tested 7 times with the kits of examples 1-3 and comparative examples 1-2 and the control CER kit, and the test results are shown in Table 6.

TABLE 62-8 deg.C storage for 0 month, 1 month, 3 months, 6 months, 18 months determination results

As can be seen from table 6, the ceruloplasmin concentrations measured at 0 month, 1 month, 3 months, 6 months and 18 months in examples 1 to 3 are close to the actual values, and the measured variation coefficients (CV values) are all less than 2%, the ceruloplasmin concentrations measured at 0 month, 1 month, 3 months, 6 months and 18 months in the control CER kit are gradually lower than the actual values (measured value at 0 month) and have a lower amplitude of more than 10%, and the ceruloplasmin concentrations measured at 0 month, 1 month, 3 months, 6 months and 18 months in comparative examples 1 to 2 are gradually lower than the actual values (measured value at 0 month) and have a lower amplitude of more than 10%.

The inventive examples differ from the comparative examples in that:

examples R1 of the present invention all used 5-30mM PB pH7.4 buffer, while comparative example 2 used 40mM PB pH7.4 buffer. The test results show that 5-30mM PB pH7.4 buffer is beneficial to stability.

② the embodiment of the invention R2 sets the optimal stabilizer proportion, while the comparative example 1 only uses NaCl as the stabilizer, the test result shows that the stability of the single stabilizer is inferior to that of the double stabilizers.

③ comparative example 1 uses the optimal 10mM PB pH7.4 buffer, but R2 is a mono-stabilizer; comparative example 2 used the optimal stabilizer ratio, but R1 was 40mM PB pH7.4 buffer. Test results show that only the optimal R1 formulation and the optimal R2 stabilizer ratio lead to optimal stability results.

3. Samples were assayed using examples 1-3 and the resulting ceruloplasmin measurements were compared to control CER kit (plain immunoturbidimetry) measurements (see figures 2-4 and regression analysis); example 1 obtained correlation r2 ═ 0.9953(y ═ 0.9566x + 0.9253); it is shown that examples 1-3 have a good correlation with the control CER kit (ordinary immunoturbidimetry).

The value of the sample measured by the control CER kit is used as an expected concentration value, the value measured by the kit of the invention is used as a measured concentration value, a plurality of samples are measured in total, and 3 groups of measured data of the plurality of samples are used for making a scatter diagram, namely the three drawings related to the invention.

Therefore, the multi-antiserum raw materials of different batches adopted in the embodiments 1-3 of the invention have better measured value correlation compared with the contrast reagent, and the batch-to-batch difference of the multi-antiserum raw materials of different batches is obviously improved in the kit of the invention.

4. The differences between the measured values of 2 different batches of the control CER kit (labeled as control kit batch 1; control kit batch 2) and the kit of the invention (example 1, example 2, and example 3 in sequence) were selected and found in FIG. 5, FIG. 6, FIG. 7, and FIG. 8.

As can be seen from FIG. 5, the control CER kit has different measurements among different batches and has poor correlation. As can be seen from FIGS. 6, 7 and 8, there was almost no difference in the assay values among the different batches of the kit of the present invention. In conclusion, the formula provided by the invention can effectively solve the problems of poor batch-to-batch stability of the CER kit.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.