CN110954692A - Reducing agent buffer solution and preparation method and application thereof - Google Patents

Abstract

The invention provides a reducing agent buffer solution and a preparation method and application thereof. The reducing agent buffer comprises: 10-100mM tris (2-carboxyethyl) phosphine, 2-10mM chelating agent and 10-100mM buffer. The tri (2-carboxyethyl) phosphine and the chelating agent in the reducing agent buffer solution are matched with each other and act coordinately, so that the influence of interference substances in a sample to be detected is reduced, the detection result is more sensitive, the specificity is higher, meanwhile, the obtained buffer solution is less influenced by temperature and concentration, the stability is better, and the accuracy of the detection result can be improved. The reducing agent buffer solution provided by the invention has the advantages of high specificity and sensitivity, stable property, simple preparation method and environmental friendliness, so that the reducing agent buffer solution can be used for preparing a detection kit, the obtained detection kit is easy to operate, and the detection result is accurate and reliable.

Description

Reducing agent buffer solution and preparation method and application thereof

Technical Field

The invention belongs to the field of biotechnological molecular detection, and particularly relates to a reducing agent buffer solution, a preparation method and application thereof, in particular to a reducing agent buffer solution for detecting a hepatitis B virus core antibody, a preparation method and application thereof.

Background

Hepatitis B (hepatitis B for short) is an infectious disease caused by Hepatitis B Virus (HBV) that can cause liver inflammation and liver function damage. Hepatitis B virus core antigen (HBcAg) is the core part of hepatitis B virus, exists in the core of Dane granule, and can stimulate human T lymphocyte to produce hepatitis B virus core antibody.

Currently, hepatitis B virus serological examination items generally comprise HBsAg, Anti-HBs, HBeAg, Anti-HBe and Anti-HBc, namely the two halves of hepatitis B commonly known as hepatitis B. HBcAg antibodies are produced shortly after onset in HBV infected persons and are often carried for life. In the HBV infection convalescent people, Anti-HBc can exist continuously, so that the Anti-HBc screening can be used for determining the previous infected individuals. The Anti-HBc assay in combination with other hepatitis B assays aids in the diagnosis and monitoring of HBV infection. In the absence of other hepatitis B markers, Anti-HBc may be the only indicator of the presence of HBV infection.

The mainstream methods for detecting Anti-HBc include enzyme-linked immunosorbent assay (ELISA), colloidal gold assay, chemiluminescence (GLIA), Electrochemiluminescence (ECL), time-resolved fluoroimmunoassay (TRFIA), and the like. Since there are a large number of specimens which are positive for Anti-HBc only in the absence of other hepatitis markers and disease symptoms, and the epidemiological relationship between the markers and the disease is not strong, erroneous judgment is likely to occur when Anti-HBc is detected. Therefore, it is important to distinguish whether a sample is a false positive sample.

In order to improve the specificity and sensitivity of a detection result, a reducing agent buffer solution is required to be used for processing a sample before the sample is detected so as to eliminate the non-specific combination of interference substances in the sample, avoid the occurrence of false positive results and improve the accuracy of the detection result.

CN102146487A discloses a hepatitis B virus nucleic acid quantitative detection kit, wherein the formulation of the solution for removing interfering substances in the kit is 1mM ethylene diamine tetraacetic acid, 0.5% SDS, 10mM Tris-HCl with pH 8.0 and 0.1% ethyl phenyl polyethylene glycol (NP-40); meanwhile, the invention adopts the real-time fluorescent quantitative PCR technology to quantitatively detect the hepatitis B virus nucleic acid, and has the advantages of specificity, sensitivity, rapidness and simple and convenient operation. However, the solution of the formula has weak oxidation resistance, and the sample is easily oxidized when the sample is processed, so that the result is inaccurate.

Therefore, the problem that the buffer solution of the reducing agent which is safe, nontoxic and stable in property and can eliminate interfering substances in a sample is needed to be solved urgently when the core antibody of the hepatitis B virus is detected is provided.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide the reducing agent buffer solution which is good in stability and environment-friendly, and the reducing agent buffer solution is good in stability, so that interfering substances in a sample can be eliminated, the specificity of an immunoreagent is improved, and the accuracy and reliability of a detection result are improved. In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a reducing agent buffer comprising: 10-100mM tris (2-carboxyethyl) phosphine, 2-10mM chelating agent and 10-100mM buffer.

The reducing agent buffer solution provided by the invention is prepared by using tris (2-carbonyl ethyl) phosphorus (TCEP), a chelating agent and the buffer solution in a reasonable proportion, wherein the TCEP has low toxicity and is easily soluble in water, the selectivity on reduction of disulfide bonds is extremely strong, and the reducing agent buffer solution prepared by using the TCEP is easier to operate when a target sample is treated; meanwhile, the buffer solution has higher stability by matching TCEP with a specific chelating agent, is less influenced by temperature and concentration, and can be used under a wider pH value range including acidic conditions (such as pH of 3), so that the hydrolysis of amido bonds is effectively reduced, and the influence of interfering substances in a target sample is reduced.

In the present invention, the concentration of tris (2-carboxyethyl) phosphine is 10 to 100mM, and may be, for example, 10mM, 15mM, 20mM, 25mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, or 100 mM.

The chelating agent may be used at a concentration of 2 to 10mM, for example, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, or 10 mM.

The buffer concentration is 10 to 100mM, and may be, for example, 10mM, 15mM, 20mM, 25mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, or 100 mM.

In a preferred embodiment of the present invention, the chelating agent is selected from one or a combination of two or more of ethylenediaminetetraacetic acid, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, citric acid, tartaric acid, and gluconic acid, preferably ethylenediaminetetraacetic acid and/or gluconic acid, and more preferably gluconic acid.

When the EDTA or the gluconic acid is used as the chelating agent, the influence of temperature and concentration on the prepared reducing agent buffer solution is smaller, which shows that when the TCEP is matched with the EDTA or the gluconic acid for use, a synergistic effect exists between the TCEP and the EDTA or the gluconic acid, and the stability of the buffer solution can be obviously improved.

Preferably, the buffer is selected from any one or a combination of more than two of Tris (hydroxymethyl) aminomethane (Tris), 4-hydroxyethylpiperazine ethanesulfonic acid (Hepes), 3- (N-morpholino) -2-hydroxypropanesulfonic acid (Mopso), 3-morpholinopropanesulfonic acid (Mops) or fatty acid Methyl Ester Sulfonate (MES).

Preferably, the pH of the reducing agent buffer is 6-8, and may be, for example, 6, 6.2, 6.4, 6.5, 6.8, 7, 7.2, 7.4, 7.5, 7.8, 7.9, or 8, and the like.

In a preferred embodiment of the present invention, the reducing agent buffer further comprises 0.1 to 200mM (for example, 0.1mM, 1mM, 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 50mM, 80mM, 100mM, 120mM, 140mM, 160mM, 180mM, 190mM, or 200 mM) of an inorganic salt.

Preferably, the inorganic salt is selected from any one or a combination of two or more of dipotassium hydrogen phosphate, potassium phosphate, sodium carbonate, potassium carbonate, sodium chloride, potassium chloride, ammonium sulfate, sodium sulfate, potassium sulfate, sodium citrate or potassium citrate.

In a preferred embodiment of the present invention, the reducing agent buffer further comprises a surfactant at a mass concentration of 0.1 to 0.5% (for example, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, or 0.5%).

Preferably, the surfactant is selected from any one or a combination of two or more of Tween (Tween), Sodium Dodecyl Sulfate (SDS) or Triton (Triton).

In a preferred embodiment of the present invention, the reducing agent buffer further comprises a preservative at a mass concentration of 0.1 to 0.5% (for example, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, or 0.5%).

Preferably, the preservative is selected from any one or a combination of more than two of Proclin300, sodium azide diazolidinyl urea, imidazolidinyl urea, sodium hydroxymethylglycine, dimethylol urea, 2-bromo-2-nitro-1, 3-diol, oxazolidine, sodium hydroxymethylglycine or adamantane quaternary ammonium.

As a preferred embodiment of the present invention, the reducing agent buffer comprises: 10-100mM tri (2-carboxyethyl) phosphine, 2-10mM ethylene diamine tetraacetic acid and/or gluconic acid, 10-100mM buffer, 0.1-200mM inorganic salt, 0.1-0.5% of surfactant by mass concentration and 0.1-0.5% of preservative by mass concentration.

In a second aspect, a method for preparing a reducing agent buffer as in the first aspect, the method comprising the steps of:

(1) mixing and homogenizing a buffering agent, a chelating agent and water according to the formula ratio to obtain a homogeneous liquid;

(2) adding tris (2-carboxyethyl) phosphine hydrochloride into the homogeneous liquid for emulsification and adjusting the pH value;

(3) adding a preservative and a surfactant, uniformly stirring, and discharging to obtain the reducing agent buffer solution.

Preferably, the temperature for mixing and homogenizing in the step (1) is 20-30 ℃, for example, 20 ℃, 22 ℃, 24 ℃, 25 ℃, 26 ℃, 28 ℃ or 30 ℃ and the like; the time is 30-45min, such as 30min, 32min, 35min, 38min, 40min, 42min or 45 min.

Preferably, the rotation speed during the mixing and homogenizing in the step (1) is 1000-.

Preferably, the rotation speed in the emulsification in the step (2) is 1000-1200rpm, such as 1000rpm, 1050rpm, 1080rpm, 1100rpm, 1120rpm, 1150rpm or 1200 rpm.

In the present invention, 6mol/L NaOH may be used to adjust the pH of the reducing agent buffer.

In a third aspect, the present invention provides a test kit comprising a reducing agent buffer as described in the first aspect.

Preferably, the detection kit is a hepatitis B virus core antibody detection kit.

The recitation of numerical ranges herein includes not only the above-recited numerical values, but also any numerical values between non-recited numerical ranges, and is not intended to be exhaustive or to limit the invention to the precise numerical values encompassed within the range for brevity and clarity.

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

(1) the reducing agent buffer solution provided by the invention is prepared by using TCEP and a chelating agent in a reasonable proportion, and the obtained buffer solution has strong oxidation resistance, is less influenced by external environments such as temperature and the like, has good stability and is convenient for long-time storage;

(2) the reducing agent buffer solution provided by the invention is simple in use method and low in toxicity, can effectively eliminate interfering substances in a sample when a target sample is processed, avoids false positive results, and obtains more accurate and reliable detection results.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

Example 1

This example provides a reducing agent buffer, and the formula of the reducing agent buffer is:

the preparation method comprises the following steps:

(1) mixing 0.02mol of MES, 0.005mol of ethylenediamine tetraacetic acid, 0.15mol of NaCl and 1000mL of water, and homogenizing to obtain a homogeneous solution;

(2) adding 0.04mol of tris (2-carboxyethyl) phosphine hydrochloride into the homogeneous liquid for emulsification and adjusting the pH value;

(3) adding 1g of a preservative Proclin300 and 5g of a surfactant Tween-20, uniformly stirring, discharging to obtain the reducing agent buffer solution, wherein the pH value of the buffer solution is 6.70.

Example 2

This example provides a reducing agent buffer obtained by leaving the reducing agent buffer of example 1 at 37 ℃ for 10 days, and the pH of the resulting buffer was 6.75.

Example 3

This example provides a reducing agent buffer obtained by leaving the reducing agent buffer of example 1 at 4 ℃ for 180 days, and the pH of the resulting buffer was 6.72.

Example 4

This example provides a reducing agent buffer, and the formula of the reducing agent buffer is:

the procedure was as in example 1, and the pH of the resulting buffer was 6.68.

Example 5

This example provides a reducing agent buffer, and the formula of the reducing agent buffer is:

the procedure was as in example 1, and the pH of the resulting buffer was 7.42.

Comparative example 1

The present comparative example provides a reducing agent buffer, the formulation of which is:

the preparation method comprises the following steps:

(1) mixing and homogenizing 0.005mol of ethylenediamine tetraacetic acid, 0.15mol of inorganic salt and 1000mL of water to obtain a homogeneous solution;

(2) adding 1g of preservative and 5g of surfactant, uniformly stirring, and discharging to obtain the reducing agent buffer solution, wherein the pH value of the obtained buffer solution is 6.72.

Comparative example 2

The procedure was as in example 1, and the pH of the resulting buffer was 6.70.

Comparative example 3

The procedure was as in example 1, and the pH of the resulting buffer was 6.70.

Comparative example 4

The procedure was as in example 1, and the pH of the resulting buffer was 6.72.

Comparative example 5

The procedure was as in example 1, and the pH of the resulting buffer was 6.68.

Performance testing

100 parts of normal human serum is taken as a detection sample, negative and positive of the detection sample are measured and counted by using examples 1-5 and comparative examples 1-5, false positive samples are rechecked for 2 times, if more than or equal to 2 positive results are judged to be false positive, and the positive results are compared with the Yapeh and Roche methodologies to reach the coincidence rate (the Yapeh detection instrument is i2000sr, and the Roche detection instrument is cobas 411).

Taking example 1 and comparative example 1 as examples, when 100 normal human sera were tested using the buffer provided in example 1 and comparative example 1, 20 samples (numbered 2, 4, 5, 8, 9, 10, 15, 20, 22, 32, 34, 46, 57, 62, 63, 66, 68, 75, 78 and 89) were found to be inconsistent, example 1 was judged to be negative, comparative example 1 was judged to be positive, and yapei and roche methodology tested the above samples to be negative, so the judgment result of example 1 was consistent with that of yapei and roche methodology, and the specific statistics are shown in table 1: (positive was judged when S/CO < 1).

TABLE 1

In examples 2-5, 100 normal human sera were used as the test samples, and the test and statistics of negative and positive results were performed, and the positive results were compared with other methods (such as yapei and roche), and found to be consistent with the results of yapei and roche tests, and no false positive results were found, indicating that the reducing agent buffer of the present invention has high accuracy and good stability.

While comparative examples 2-5 all showed false positive results: comparative example 2 false positive results were obtained for 2 samples (nos. 4 and 32) tested, with hbcabs S/CO means of 0.85 and 0.91, respectively; thus, as can be seen from a comparison of example 1 with comparative example 2, the TCEP used in the present invention enables a higher accuracy of the reducing agent buffer compared to DTT.

In comparative example 3, 8 samples (nos. 4, 22, 32, 46, 62, 68 and 89) were tested for false positive results, and the HBcAbS/CO mean values were 0.75, 0.84, 0.81, 0.77, 0.86, 0.92 and 0.89; therefore, as can be seen from the comparison between example 1 and comparative example 3, the TCEP adopted in the present invention is coordinated with ethylenediaminetetraacetic acid, so that the accuracy of the detection result can be significantly improved.

In the test of comparative example 4, 5 samples (numbered 4, 32, 46, 68 and 89) showed false positive results, and the HBcAb S/CO mean values were 0.82, 0.87, 0.91, 0.95 and 0.84; in comparative example 5, false positive results were observed in 2 samples (numbers 32 and 46) and the HBcAb S/CO mean values were 0.87 and 0.89, respectively. Therefore, as can be seen from the comparison of example 1 with comparative examples 4 and 5, the accuracy is also reduced when the TCEP and EDTA contents in the solution are outside the range of the present invention.

In conclusion, the reducing agent buffer solution provided by the invention can well reduce nonspecific reactions, improve the specificity of reagents and reduce the false positive probability of clinical sample detection, and meanwhile, the reducing agent buffer solution provided by the invention has good stability and can be stored for a long time.

The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.

Claims (10)

1. A reducing agent buffer, wherein the reducing agent buffer comprises: 10-100mM tris (2-carboxyethyl) phosphine, 2-10mM chelating agent and 10-100mM buffer.

2. The reducing agent buffer according to claim 1, wherein the chelating agent is selected from any one or a combination of two or more of ethylenediaminetetraacetic acid, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, citric acid, tartaric acid, or gluconic acid, preferably ethylenediaminetetraacetic acid and/or gluconic acid, and more preferably gluconic acid;

preferably, the buffer is selected from any one or the combination of more than two of trihydroxymethyl aminomethane, 4-hydroxyethyl piperazine ethanesulfonic acid, 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid salt, 3-morpholinyl propanesulfonic acid or fatty acid methyl ester sulfonate;

preferably, the pH of the reducing agent buffer is 6-8.

3. The reducing agent buffer according to claim 1 or 2, characterized in that the reducing agent buffer further comprises 0.1-200mM of an inorganic salt;

preferably, the inorganic salt is selected from any one or a combination of two or more of dipotassium hydrogen phosphate, potassium phosphate, sodium chloride, potassium chloride, ammonium sulfate, sodium sulfate, potassium sulfate, sodium citrate or potassium citrate.

4. The reducing agent buffer according to any one of claims 1 to 3, further comprising a surfactant at a mass concentration of 0.1 to 0.5%;

preferably, the surfactant is selected from any one or a combination of more than two of tween, sodium dodecyl sulfate or Triton.

5. The reducing agent buffer according to any one of claims 1 to 4, further comprising a preservative at a mass concentration of 0.1 to 0.5%;

preferably, the preservative is selected from any one or a combination of more than two of Proclin300, sodium azide diazolidinyl urea, imidazolidinyl urea, sodium hydroxymethylglycine, dimethylol urea, 2-bromo-2-nitro-1, 3-diol, oxazolidine, sodium hydroxymethylglycine or adamantane quaternary ammonium.

6. The reducing agent buffer according to any one of claims 1 to 5, wherein the reducing agent buffer comprises:

10-100mM tri (2-carboxyethyl) phosphine, 2-10mM ethylene diamine tetraacetic acid and/or gluconic acid, 10-100mM buffer, 0.1-200mM inorganic salt, 0.1-0.5% of surfactant by mass concentration and 0.1-0.5% of preservative by mass concentration.

7. A method for preparing a reducing agent buffer according to claims 1 to 6, comprising the steps of:

(1) mixing and homogenizing a buffering agent, a chelating agent and water according to the formula ratio to obtain a homogeneous liquid;

(2) adding tris (2-carboxyethyl) phosphine hydrochloride into the homogeneous liquid for emulsification and adjusting the pH value;

(3) adding a preservative and a surfactant, uniformly stirring, and discharging to obtain the reducing agent buffer solution.

8. The preparation method according to claim 7, wherein the temperature of the mixing and homogenizing in the step (1) is 20-30 ℃ and the time is 30-45 min;

preferably, the rotation speed of the mixing and homogenizing in the step (1) is 1000-;

preferably, the rotation speed in the emulsification in the step (2) is 1000-1200 rpm.

9. A test kit comprising the reducing agent buffer of any one of claims 1 to 6.

10. The detection kit according to claim 9, wherein the detection kit is a hepatitis b virus core antibody detection kit.