CN114778816A - Acridinium ester labeling compound and preparation method thereof - Google Patents

Abstract

The invention discloses an acridinium ester labeling compound which comprises a body and a labeling group labeled on the body, wherein the body is an antibody, and the labeling group is an acridinium ester group. A preparation method of an acridinium ester labeling compound comprises the following steps: absorbing the labeled buffer solution into a centrifugal tube; sucking an antibody to be labeled into a centrifuge tube and uniformly mixing to generate a first mixed solution; adding the acridine ester solution, uniformly mixing, and reacting for 30 minutes at 37 ℃ in the dark to generate a second mixed solution; adding a termination buffer solution, mixing uniformly, and reacting for 10 minutes at 37 ℃ in a dark place to generate a third mixed solution; desalting and purifying the third mixed solution to generate a fourth mixed solution; and adding a protein diluent into the fourth mixed solution to generate the acridinium ester labeled complex. According to the preparation method, the labeling efficiency of the acridinium ester and the antibody is higher, and the reaction time is short; the nonspecific adsorption is lower, the sensitivity is higher, and the labeling method is simple, quick and easy to repeat.

Description

Acridinium ester labeling compound and preparation method thereof

Technical Field

The invention belongs to the field of biotechnology detection, and particularly relates to an acridinium ester labeled compound and a preparation method thereof.

Background

Chemiluminescence immunoassay (CLIA) is a detection and analysis technique for various antigens, haptens, antibodies, hormones, drugs and the like by combining a chemiluminescence assay technique with high sensitivity and high specificity immunoreaction. The method is an ultra-high-sensitivity determination technology developed after radioimmunoassay, enzyme immunoassay, fluorescence immunoassay and time-resolved fluorescence immunoassay.

Chemiluminescent immunoassays are generally classified into two types, direct light emission and indirect light emission. Indirect luminescence is also called enzymatic luminescence, the duration of luminescence is long, the luminescence belongs to a glow type, and the markers of the luminescence are Horse Radish Peroxidase (HRP) and Alkaline Phosphatase (AP); the direct luminescence belongs to a flash type, the luminescence time is short, and the main markers are acridinium ester, isoluminol, terpyridyl ruthenium labeled electrochemical luminescence and the like. The indirect luminescent system is easily influenced by factors such as environmental pH value, temperature and the like due to the participation of enzyme, the preparation of luminescent substrate is complex, and the reaction background value is high; the terpyridyl ruthenium electrochemiluminescence system has higher requirements on the performance of instruments and equipment and also has strict requirements on cleaning conditions; compared with other chemiluminescence systems, the acridinium ester chemiluminescence system has the advantages of low background, high labeling efficiency, short luminescence time, good stability and the like, and is widely applied to in vitro diagnosis technology at present.

The acridinium ester chemiluminescence is of a flash type, has advantages compared with other technologies in the field of chemiluminescence immunoassay, reaches maximum light intensity after 0.4s of the addition of a starter, has a half-life period of 0.9s, basically finishes luminescence within 2s, and can realize rapid detection. The commonly used labeling substance of the system is acridinium ester, acridinium sulfonamide or the like, the principle of labeling with an antibody is not complex, but because the acridinium ester is usually dissolved in an organic solvent, such as dimethyl sulfoxide (DMSO), the contact with antibody protein can cause the risk of protein denaturation or activity reduction, the luminous efficiency of the acridinium ester is extremely high, and the residual acridinium ester can cause abnormal states such as high reaction background value, high nonspecific reaction, poor repeatability and the like.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide an acridinium ester labeling compound and a preparation method thereof, aiming at solving the problem that the residual organic solvent for dissolving acridinium ester in the prior art is contacted with antibody protein to cause the risk of protein denaturation or activity reduction; and the residual free acridinium ester and non-target products can cause the problems of high reaction background value, high non-specific reaction, poor repeatability and other abnormal states.

The invention is realized by adopting the following technical scheme:

the acridinium ester labeling compound comprises a body and a labeling group labeled on the body, wherein the body is an antibody, and the labeling group is an acridinium ester group.

In order to optimize the technical scheme, the specific measures adopted further comprise:

further, the antibody is a troponin T antibody, a thyroid stimulating hormone antibody, or a triiodothyronine antibody.

Further, the acridine ester group comprises acridine ester or acridine sulfonamide.

A preparation method of an acridinium ester labeled complex comprises the following steps:

absorbing a marking buffer solution into a centrifuge tube, wherein the pH value of the marking buffer solution is more than 7;

sucking an antibody to be labeled into a centrifugal tube and uniformly mixing to generate a first mixed solution;

adding the acridine ester solution, uniformly mixing, and reacting for 30 minutes at 37 ℃ in the dark to generate a second mixed solution;

adding a termination buffer solution, mixing uniformly, and reacting for 10 minutes at 37 ℃ in a dark place to generate a third mixed solution;

desalting and purifying the third mixed solution to generate a fourth mixed solution;

and adding a protein diluent into the fourth mixed solution to generate an acridinium ester labeled compound, and storing at the temperature of 2-8 ℃ in a dark place.

Further, the labeling buffer solution is 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution, phosphate buffer solution, carbonate buffer solution and 2- (N-morpholine) ethanesulfonic acid.

Further, the concentration of the antibody in the first mixed solution was 1 mg/ml.

Further, the mixing ratio of the acridinium ester solution to the antibody is 10-80: 1.

further, the mass of the termination buffer is not less than 10 times of the mass of the antibody, and the termination buffer is a lysine solution with the concentration of not less than 10 mg/L.

Further, desalting and purifying the third mixed solution to generate a fourth mixed solution specifically comprises the following steps:

desalting and primarily purifying by using an elution buffer solution to remove small molecular impurities and free acridine ester, wherein the elution buffer solution is 0.1mol/L phosphate buffer solution;

and purifying the primarily purified third mixed solution again by using a filter membrane to remove by-products generated in the reaction process of the acridinium ester and the antibody, so as to generate a fourth mixed solution.

The invention has the beneficial effects that:

compared with the prior art, the acridinium ester labeling compound and the preparation method thereof have the advantages that the labeling efficiency of the acridinium ester and an antibody is higher, and the reaction time is short; the nonspecific adsorption is lower, the sensitivity is higher, and the marking method is simple, quick and easy to repeat, and is convenient for large-scale production and use; based on the method, the chemiluminescence immunoassay detection kit which has higher sensitivity, wider linear range, meets the requirement of clinical use and is convenient to realize automation can be prepared.

Drawings

FIG. 1 is a diagram of the structure of acridinium ester sulfonamide molecules of the present invention.

FIG. 2 is a diagram showing the structure of an acridinium ester molecule of the present invention.

FIG. 3 is a flow chart of the preparation of acridinium ester labeled complexes of the present invention.

FIG. 4 is a comparison of the test results of the kit prepared from the acridinium ester antibody and the commercial kit in the embodiment 1 of the present invention.

FIG. 5 is a comparison of the test results of the kit prepared from the acridinium ester antibody and the commercial kit in the embodiment 2 of the present invention.

FIG. 6 is a comparison chart of the test results of the kit prepared from the acridinium ester antibody and the test results of the commercialized kit in embodiment 3 of the present invention.

Detailed Description

In order to clarify the technical solutions and operating principles of the present invention, the present invention is further described in detail with reference to specific embodiments in the following drawings, and it should be noted that, without conflict, any combination between the embodiments described below or between the technical features may form a new embodiment.

The invention provides an acridinium ester labeling compound which comprises a body and a labeling group labeled on the body, wherein the body is an antibody, and the labeling group is an acridinium ester group.

Wherein the antibody is troponin T antibody, thyroid stimulating hormone antibody, triiodothyronine antibody; the marking group is an acridine ester compound; the acridine ester compounds comprise acridine ester and acridine sulfonamide.

As shown in fig. 3, a method for preparing an acridinium ester labeled complex is provided, which is characterized by comprising the following steps:

s1: absorbing a marking buffer solution into a centrifuge tube, wherein the pH value of the marking buffer solution is more than 7;

specifically, the method comprises the following steps: the marking buffer solution is 4-ethoxyl piperazine ethanesulfonic acid buffer solution, phosphate buffer solution, carbonate buffer solution and 2- (N-morpholine) ethanesulfonic acid. Different buffer concentrations and pH values affect the labeling efficiency of the antibody. A preferred buffer for use in the present invention is HEPES buffer at pH8.0, and 0.05 mol/L.

S2: sucking an antibody to be labeled into a centrifugal tube and uniformly mixing to generate a first mixed solution; the concentration of the antibody in the first mixed solution is 1 mg/ml;

specifically, the method comprises the following steps: since acridinium ester is stored in an organic solvent, the organic solvent affects the activity of labeled antibody, but if the organic solvent is diluted too much to simply reduce the content of the organic solvent, the concentration of antibody and the concentration of acridinium ester are reduced simultaneously, and the labeling efficiency is reduced, and the antibody concentration is preferably 1 mg/ml.

S3: adding the acridine ester solution, uniformly mixing, and reacting for 30 minutes at 37 ℃ in the dark to generate a second mixed solution;

specifically, the method comprises the following steps: the acridinium ester is a derivative with an acridinium ring structure, and comprises the acridinium ester, acridine sulfonamide and the like, the structural formula is shown in figure 1 and figure 2, and the addition molar ratio of the acridinium ester to the antibody to be labeled is (10-80): 1. the different marking ratios have a great influence on the result but need to be determined according to specific examples; the length of the reaction time affects the labeling efficiency of the antibody and the intensity of the luminescent signal, and an excessively long reaction time also has a certain influence on the activity of the antibody. The preferred reaction time for the present invention is 30 minutes.

S4: adding a termination buffer solution, mixing uniformly, and reacting for 10 minutes at 37 ℃ in a dark place to generate a third mixed solution;

specifically, the method comprises the following steps: the termination buffer solution is a lysine solution with the concentration not lower than 10mg/L, and the mass of the termination buffer solution is not lower than 10 times of the mass of the antibody.

S5: desalting and purifying the third mixed solution to generate a fourth mixed solution;

specifically, the method comprises the following steps: the by-products generated in the reaction process of the acridinium ester and the antibody, such as the failure of effective removal, can cause problems of higher background value, and poor repeatability caused by uncertain nonspecific adsorption. The desalting and purifying operation is carried out in the step, so that the test background value of the labeled acridinium ester antibody is lower and the repeatability is better compared with the conventional method.

S51: desalting and primarily purifying by using an elution buffer solution to remove small molecular impurities, wherein the elution solution is 0.1mol/L phosphate buffer solution;

wherein, desalting purification can adopt desalting column preloaded with G25 packing to remove free small molecule acridinium ester.

S52: and purifying the primarily purified third mixed solution again by using a filter membrane to remove by-products generated in the reaction process of the acridinium ester and the antibody, thereby generating a fourth mixed solution.

Specifically, the method comprises the following steps: step S51 can remove small molecular impurities in the solution quickly, but in the coupling reaction of the antibody and acridinium ester, a small amount of antibody will change conformation due to denaturation, and meanwhile, the general antibody contains a certain amount of hetero-protein (the purity of the antibody is about 95%), and the reactant generated after the reaction of the two substances and acridinium ester has uncertain non-specific adsorption on the subsequent immune reaction, which results in poor repeatability and affects the detection performance of the reagent. The removal of such substances cannot be performed by desalting or dialysis because of their large molecular weight and the molecular weight of the added substances subjected to protein denaturation and side reaction is larger than that of the target product, and the processes such as liquid phase separation require special equipment and are expensive and complicated. Adopt the filter membrane of corresponding specification to filter in this scheme, consequently utilize the specification to be aperture 0.45um through preferred, the material of filter membrane includes but not limited to nylon, cellulose nitrate, cellulose acetate etc..

S6: and adding a protein diluent into the fourth mixed solution to generate an acridinium ester labeled compound, and storing at 2-8 ℃ in a dark place.

Detailed description of the preferred embodiment 1

Taking the example of marking 0.1mg of troponin T (CTNT) antibody, the preferred marking ratio of the example is 40:1, and the specific steps are as follows:

0.1mg of CTNT antibody was aspirated, diluted to 1mg/ml with a labeling buffer (HEPES pH 8.0), and mixed well;

adding the acridinium ester solution according to the labeling proportion, fully and uniformly mixing, and carrying out a light-shielding reaction at 37 ℃ for 30 minutes;

after the reaction is finished, 100ul of lysine solution with the concentration of 10mg/ml is added, the mixture is fully and uniformly mixed, and the mixture is subjected to light-resistant reaction at 37 ℃ for 10 minutes;

after the reaction is finished, eluting and purifying by using a G25 desalting column, wherein the elution buffer solution is 0.1mol/L PBS buffer solution;

after the elution is finished, the filtration device is used for filtration and purification again in combination with a filter membrane with the pore diameter of 0.45 um;

after the filtration, 100ul of 10% BSA solution is added and mixed, and the mixture is stored at 2-8 ℃ in a dark place.

Experimental comparison results:

table-linear testing meter

Second blank background value test meter

As shown in the table I and the table II, the table I is a linear test table, the table II is a blank background value test table, and compared with the conventional method, the linearity and blank limits are tested, and the test result shows that the acridinium ester antibody prepared by the invention has better low-value linearity and lower background value.

As shown in FIG. 4, the concentration one is the concentration of the sample detected by the kit prepared based on the acridinium ester antibody labeled in embodiment 1; the kit with the second concentration (Roche diagnosis) is used for detecting the concentration of the sample, and the correlation coefficient R is more than or equal to 0.99 through comparison, so that the kit has excellent correlation with the commercial kit. The kit prepared by the technical scheme has high sensitivity, the detection and analysis capability of low-value samples is improved qualitatively, and the clinical significance of the item is combined, so that the kit is in line with the use of clinical auxiliary diagnosis. In conclusion, the test data analysis shows that the acridinium ester antibody marked by the invention has higher test sensitivity and better comparison correlation with a commercialized kit, and meets the clinical detection requirement.

Specific embodiment example 2:

taking the example of labeling 0.1mg Thyroid Stimulating Hormone (TSH) antibody, the preferred labeling ratio of this example is 20:1, and the specific steps are as follows:

0.1mg of TSH antibody was aspirated and diluted to 1mg/ml with a labeling buffer (HEPES pH 8.0), followed by thorough mixing;

adding the acridinium ester solution according to the labeling proportion, fully and uniformly mixing, and carrying out a light-shielding reaction at 37 ℃ for 30 minutes;

after the reaction is finished, 100ul of lysine solution with the concentration of 10mg/ml is added, the mixture is fully and uniformly mixed, and the mixture is subjected to light-resistant reaction at 37 ℃ for 10 minutes;

after the reaction is finished, eluting and purifying by using a G25 desalting column, wherein the elution buffer solution is 0.1mol/L PBS buffer solution;

after the elution is finished, the mixture is filtered and purified again by using a filtering device combined with a filter membrane with the pore diameter of 0.45 um;

after the filtration, 100ul of 10% BSA solution is added and mixed evenly, and the mixture is stored in the dark at the temperature of 2-8 ℃.

Experimental comparison results:

comparison table for effect of filter membrane

Test meter for testing four-repeatability of meter

As shown in the third table and the fourth table, the by-products generated in the reaction process of the acridinium ester and the antibody can be filtered greatly by adopting the filter membrane with the pore diameter of 0.45um for secondary filtration and purification, wherein CV represents repeatability, and experiments show that the background value of the acridinium ester labeled compound prepared by the invention is lower and the repeatability is fundamentally improved.

As shown in FIG. 5, the concentration one is the concentration of the sample detected by the kit prepared based on the acridinium ester antibody labeled in embodiment 2; the kit with the second concentration (Roche diagnosis) is used for detecting the sample concentration, and through comparison, the correlation coefficient R is more than or equal to 0.99, so that the kit has excellent correlation with the commercial kit and meets the clinical detection requirement. Based on data analysis of the test by the method, the test sensitivity of the kit prepared by using the labeled acridinium ester antibody is higher, the background value is lower, the nonspecific adsorption is obviously reduced, and particularly, the repeatability is fundamentally changed. Compared with a commercialized kit, the kit has better correlation and meets the clinical detection requirement.

Specific embodiment example 3:

taking the example of labeling 0.1mg of tetraiodothyronine (T4) antibody, the preferred labeling ratio of the example is 40:1, and the specific steps are as follows:

0.1mg of T4 antibody was pipetted and diluted to 1mg/ml with a labeling buffer (HEPES pH 8.0), and mixed well;

adding the acridinium ester solution according to the labeling proportion, fully and uniformly mixing, and carrying out a light-resistant reaction at 37 ℃ for 30 minutes;

after the reaction is finished, 100ul of lysine solution with the concentration of 10mg/ml is added, the mixture is fully and uniformly mixed, and the mixture is subjected to light-resistant reaction at 37 ℃ for 10 minutes;

after the reaction is finished, eluting and purifying by using a G25 desalting column, wherein the elution buffer solution is 0.1mol/L PBS buffer solution;

after the elution is finished, the filtration device is used for filtration and purification again in combination with a filter membrane with the pore diameter of 0.45 um;

after the filtration, 100ul of 10% BSA solution is added and mixed evenly, and the mixture is stored in the dark at the temperature of 2-8 ℃.

The result of comparison between the test results of the kit (competitive method) prepared based on the labeled acridinium ester antibody and the test results of the commercialized kit meets the requirement of clinical detection.

Experimental comparison results:

watch five repeatability detection watch

As shown in table five and fig. 6, the first concentration is the concentration of the sample detected by the kit prepared based on the acridinium ester antibody labeled in embodiment 3; the kit with the second concentration (Roche diagnosis) is used for detecting the sample concentration, both are detected by a competition method, and through comparison, the correlation coefficient R is more than or equal to 0.9, so that the kit has better correlation with the commercial kit and meets the clinical detection requirement. Based on the data analysis of the test by the method, the test repeatability of the kit prepared by using the labeled acridinium ester antibody meets the clinical requirement, and meanwhile, the kit has better correlation with a commercialized kit, and meets the clinical detection requirement.

The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above-mentioned embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may be made by those skilled in the art without departing from the principle of the invention.

Claims (9)

1. The acridinium ester labeling compound is characterized by comprising a body and a labeling group labeled on the body, wherein the body is an antibody, and the labeling group is an acridinium ester group.

2. The acridinium ester labeling complex of claim 1, wherein: the antibody is a troponin T antibody, a thyroid stimulating hormone antibody or a triiodothyronine antibody.

3. The acridinium ester labeling complex of claim 1, wherein: the acridine ester group comprises acridine ester or acridine sulfonamide.

4. The preparation method of the acridinium ester labeled complex is characterized by comprising the following steps of:

absorbing a marking buffer solution into a centrifuge tube, wherein the pH value of the marking buffer solution is more than 7;

sucking an antibody to be labeled into a centrifugal tube and uniformly mixing to generate a first mixed solution;

adding the acridine ester solution, uniformly mixing, and reacting for 30 minutes at 37 ℃ in the dark to generate a second mixed solution;

adding a termination buffer solution, mixing uniformly, and reacting for 10 minutes at 37 ℃ in a dark place to generate a third mixed solution;

desalting and purifying the third mixed solution to generate a fourth mixed solution;

and adding a protein diluent into the fourth mixed solution to generate an acridinium ester labeled complex.

5. The method for preparing an acridinium ester labeling complex according to claim 4, wherein: the marking buffer solution is one or more of 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution, phosphate buffer solution, carbonate buffer solution and 2- (N-morpholine) ethanesulfonic acid.

6. The method for preparing an acridinium ester labeling complex according to claim 4, wherein: the concentration of the antibody in the first mixed solution was 1 mg/ml.

7. The method for preparing an acridinium ester labeling complex according to claim 4, wherein: the molar ratio of the acridinium ester solution to the antibody is 10-80: 1.

8. the method for preparing an acridinium ester labeling complex according to claim 4, wherein: the mass of the termination buffer solution is not less than 10 times of the mass of the antibody, and the termination buffer solution is a lysine solution with the concentration of not less than 10 mg/L.

9. The method for preparing an acridinium ester labeling complex according to claim 4, wherein: desalting and purifying the third mixed solution to generate a fourth mixed solution, wherein the process for generating the fourth mixed solution specifically comprises the following steps:

desalting and primarily purifying by using an elution buffer solution to remove small molecular impurities and free acridine ester, wherein the elution buffer solution is 0.1mol/L phosphate buffer solution;

and purifying the primarily purified third mixed solution again by using a filter membrane to remove by-products generated in the reaction process of the acridinium ester and the antibody, so as to generate a fourth mixed solution.

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