CN115932284A - Method for coupling acridine ester with immune protein and application thereof - Google Patents
Method for coupling acridine ester with immune protein and application thereof Download PDFInfo
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Abstract
The invention discloses a method for coupling acridinium ester with immune protein and application thereof, relating to the technical field of immunoassay, wherein the method comprises the steps of dissolving acridinium ester in aprotic anhydrous N, N-dimethylformamide or anhydrous dimethyl sulfoxide added with methylated-beta-cyclodextrin before the acridinium ester is coupled with the immune protein, wherein the concentration of the acridinium ester is 5mg/mL, and the molar concentration ratio of the methylated-beta-cyclodextrin to the acridinium ester is 0.5:1-2:1; compared with the prior art, the acridinium ester coupled immunoprotein treated by the method has the advantages that the stability and the luminous efficiency of the acridinium ester are effectively improved, and the problems that the acridinium ester is not free from illumination and heating in the process of storage or coupling in the prior art and the stability and the luminous efficiency are difficult to ensure because the acridinium ester needs to participate in a reaction in a water-soluble medium are solved.
Description
Technical Field
The invention relates to the technical field of immunoassay, in particular to a method for coupling acridinium ester with immune protein and application thereof.
Background
Chemiluminescence immunoassay combines a chemiluminescence assay technology with high sensitivity and high specificity immunoreaction, and is used for detection and analysis technologies of various antigens, haptens, antibodies, hormones, medicines and the like. 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 horseradish peroxidase and alkaline phosphatase; the direct luminescence belongs to a flash type, the luminescence time is short, and the main markers are acridinium ester, isoluminol, electrochemical luminescence marked by terpyridyl ruthenium 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 is yellow powder in appearance, is yellow green liquid after being dissolved, is an important chemiluminescent reagent, has higher quantum yield and higher chemiluminescent efficiency, and is usually five times or more than five times of luminol. The specific light-emitting principle is that in an alkaline hydrogen peroxide solution, acridinium ester is attacked by hydrogen peroxide ions to generate unstable dioxyethane with tension, the unstable dioxyethane is further decomposed into carbon dioxide and acridone in an electronic excited state, and when the acridone returns to a ground state, photons with the maximum absorption wavelength of 430nm are emitted. This luminescence process is very short (the whole process takes place in less than 2 seconds). Proteins, polypeptides, antibodies, nucleic acids may be labelled with the product.
Before the acridine ester is coupled with protein, because the terminal carboxyl of the acridine ester is activated by N-hydroxysuccinimide and is active, the acridine ester needs to be dissolved by an aprotic dry anhydrous solvent, and besides anhydrous N, N-dimethylformamide, the acridine ester can also be dissolved by an aprotic solvent such as anhydrous dimethyl sulfoxide and the like. After protein coupling, acridine ester exists between the acridine ester and a labeled substance in the forms of amido bonds or ester bonds and the like, the active site of the acridine ester is unstable in alkaline and oxidizing environments, the stability of the acridine ester coupling compound is reduced due to partial hydrolysis, and the hydrolysis process is a non-luminous dark reaction process; the degree of hydrolysis increases with increasing alkalinity and with increasing temperature. The acridinium ester and the coupling compound thereof are likely to be partially decomposed under the condition of illumination so as to influence the luminous effect, the experimental operation and storage of the acridinium ester suggest treatment under the condition of avoiding light, and at present, no specific research data exists temporarily. The prior art CN202111616411.X (acridinium ester labeled antibody and a preparation method and application thereof) discloses that the acridinium ester labeled antibody utilizes polyamino auricularia auricula polysaccharide AF1 to connect acridinium ester and an antibody, so that the combination amount of the acridinium ester on the antibody can be improved, the detection sensitivity is further improved, meanwhile, the polyamino auricularia auricula polysaccharide AF1 cannot cause excessive obstruction to the combination between the acridinium ester labeled antibody and an antigen, and the improvement of the detection sensitivity is facilitated; the prior art CN202210359050.3 (an acridinium ester labeled compound and a preparation method) discloses a preparation method of an acridinium ester labeled compound, which solves the risks of protein denaturation or activity reduction caused by the contact of acridinium ester dissolved in an organic solvent and antibody protein by a method for controlling the final concentration of antibody reaction, and solves the problems of abnormal states such as high reaction background value, high nonspecific reaction, poor repeatability and the like caused by residual acridinium ester by a method for purifying twice. However, the problem that the acridine ester and the coupling compound thereof are possibly partially decomposed under the illumination condition is not mentioned, the experimental operation process of the acridine ester is difficult to completely avoid light, the repeatability and the stability of the process are influenced to a great extent, and the problem needs to be solved urgently.
Disclosure of Invention
The invention firstly provides a method for coupling acridinium ester with immune protein, which comprises the following steps: before the acridine ester is coupled with the immune protein, the acridine ester is dissolved in aprotic anhydrous N, N-dimethylformamide or anhydrous dimethyl sulfoxide added with methylated-beta-cyclodextrin and mixed.
The invention also provides a method for coupling the acridinium ester with the immunoprotein, which comprises the following steps:
step (1): dialyzing the immune protein;
step (2): absorbing the labeled buffer solution into a centrifuge tube to generate a solution 1;
and (3): sucking dialyzed immune protein into a centrifuge tube and mixing uniformly to generate a solution 2;
and (4): adding the acridine ester solution, uniformly mixing, and slowly oscillating at the temperature of 25 +/-2 ℃ for light-shielding reaction for 2 hours to generate a solution 3;
and (5): desalting and purifying the solution 3 to generate a solution 4;
and (6): adding an acridinium ester preservation buffer solution into the solution 4 to generate an acridinium ester coupled immune protein complex solution, and preserving at the temperature of 2-8 ℃ in a dark place.
In one embodiment according to the present invention, the immunity protein in step (1) comprises an antibody and an antigen, specifically, the antibody is murine immunoglobulin G, and the antigen mainly comprises 25-hydroxy-vitamin D-bovine serum albumin conjugate;
the dialysate adopts a labeling buffer solution, and the labeling buffer solution is a phosphoric acid buffer solution with the pH value of 8.0 +/-0.2;
in one embodiment according to the present invention, step (4): adding the acridine ester solution, uniformly mixing, and slowly oscillating at the temperature of 25 +/-2 ℃ for light-shielding reaction for 2 hours to generate a solution 3;
the acridinium ester used for coupling comprises acridinium salt NSP-DMAE-NHS or acridinium salt NSP-SA-NHS;
dissolving acridine ester in aprotic anhydrous N, N-dimethylformamide or anhydrous dimethyl sulfoxide added with methylated-beta-cyclodextrin, wherein the concentration of the acridine ester is 5mg/mL, and the molar concentration ratio of the methylated-beta-cyclodextrin to the acridine ester is 0.5:1-2:1;
diluting the dissolved acridinium ester with a labeling buffer solution until the volume of the dissolved acridinium ester is consistent with that of the solution 2;
the molar ratio of the acridinium ester to the immunoprotein is: 10, 1 to 50;
after fully mixing, slowly oscillating and reacting for 2 hours in the dark at 25 plus or minus 2 ℃ to generate a solution 3.
In one embodiment according to the present invention, step (5) further comprises purification using a G50/G25 chromatography column to separate excess unbound acridinium ester, methylated- β -cyclodextrin, and acridinium ester-coupled immune protein complex.
In one embodiment according to the present invention, step (6) further comprises diluting the collected acridinium ester-conjugated immunoprotein complex to 0.01ug/mL with acridinium ester preservation buffer for detection.
In yet another aspect, the invention provides the use of methylated- β -cyclodextrin in the preparation of a reagent for increasing the luminescent signal and efficiency of an acridinium ester-coupled immunoprotein immunoassay.
The technical scheme of the invention has the following beneficial effects:
the acridinium ester is generally kept or coupled without the problems of illumination and heating, and needs to participate in reaction in a water-soluble medium, so that the stability and luminous efficiency of the acridinium ester are difficult to ensure in the prior art.
By adding the methylated-beta-cyclodextrin into the acridinium ester preservative solution, the stability and the luminous efficiency of the acridinium ester are effectively improved. The possible reason is that the methylated-beta-cyclodextrin has a very peculiar spatial structure, namely a cylindrical hollow structure, and partial groups of the acridinium ester can be enveloped in the cylindrical structure, so that the photo-thermal stability of the acridinium ester is improved, and meanwhile, the coupling reaction of the residual groups and the antibody is not influenced.
Drawings
FIG. 1 is a flow diagram of acridinium ester-coupled immunity proteins;
FIG. 2 is a comparison of luminescence signals of acridinium ester-coupled immune protein complex solutions;
FIG. 3 is a comparison of luminescence signals of different acridinium ester-conjugated antibody complex solutions;
FIG. 4 is a comparison of luminescence signals of different acridinium ester-conjugated antigen complex solutions.
Detailed Description
In order to make the technical problems, technical solutions and advantages of the present invention more apparent, the following detailed description is given with reference to the accompanying drawings and specific embodiments.
Example 1 procedure for coupling acridinium esters to immunoproteins
The first step is as follows: dialyzing the immune protein;
the immune protein comprises an antibody and an antigen, specifically, the antibody is murine immunoglobulin G, and the antigen mainly comprises a 25-hydroxy-vitamin D-bovine serum albumin conjugate;
the dialysate adopts a labeling buffer solution, and the labeling buffer solution is a phosphoric acid buffer solution with the pH value of 8.0 +/-0.2;
the second step is that: absorbing the labeled buffer solution into a centrifuge tube to generate a solution 1;
the third step: sucking dialyzed immune protein into a centrifuge tube and mixing uniformly to generate a solution 2;
the concentration of the antibody in the solution 2 is 1-2mg/mL, and the concentration of the antigen is 0.5-1mg/mL;
the fourth step: adding the acridinium ester solution, uniformly mixing, and slowly oscillating and keeping away from light for 2 hours at the temperature of 25 +/-2 ℃ to generate a solution 3;
the acridinium ester used for coupling comprises acridinium salt NSP-DMAE-NHS or acridinium salt NSP-SA-NHS;
dissolving acridine ester in aprotic anhydrous N, N-dimethylformamide or anhydrous dimethyl sulfoxide added with methylated-beta-cyclodextrin, wherein the concentration of the acridine ester is 5mg/mL, and the molar concentration ratio of the methylated-beta-cyclodextrin to the acridine ester is 0.5:1-2:1;
the acridine ester is firstly dissolved in aprotic anhydrous N, N-dimethylformamide or anhydrous dimethyl sulfoxide added with beta-cyclodextrin, the concentration is 5mg/mL, and the molar concentration ratio of the beta-cyclodextrin to the acridine ester is 0.5:1-2:1;
diluting the dissolved acridinium ester with a labeling buffer solution until the volume of the dissolved acridinium ester is consistent with that of the solution 2;
the molar ratio of the acridinium ester to the immunoprotein is: 10, 1 to 50;
after fully mixing, slowly oscillating and reacting for 2 hours in the dark at 25 plus or minus 2 ℃ to generate a solution 3.
The fifth step: desalting and purifying the solution 3 to generate a solution 4;
purifying with G50/G25 chromatographic column to separate excess unbound acridinium ester, methylated-beta-cyclodextrin (or beta-cyclodextrin), and acridinium ester-coupled immune protein complex;
and a sixth step: adding an acridinium ester preservation buffer solution into the solution 4 to generate an acridinium ester coupled immune protein complex solution, and preserving at the temperature of 2-8 ℃ in a dark place.
The collected acridinium ester coupling immune protein compound is diluted to 0.01ug/mL by acridinium ester storage buffer solution for detection.
Example 2 stability and homogeneity monitoring of acridinium esters
Preparation of acridine ester solution 1: dissolving the acridine salt NSP-SA-NHS in anhydrous dimethyl sulfoxide, wherein the concentration of the acridine ester is 5mg/mL;
preparation of acridine ester solution 2: dissolving an acridine salt NSP-SA-NHS in anhydrous dimethyl sulfoxide added with methylated-beta-cyclodextrin, wherein the concentration of acridine ester is 5mg/mL, and the molar concentration ratio of the methylated-beta-cyclodextrin to the acridine ester is 0.5:1;
preparation of acridine ester solution 3: dissolving an acridine salt NSP-SA-NHS in anhydrous dimethyl sulfoxide added with methylated-beta-cyclodextrin, wherein the concentration of acridine ester is 5mg/mL, and the molar concentration ratio of the methylated-beta-cyclodextrin to the acridine ester is 2:1;
preparation of acridine ester solution 4: dissolving acridine salt NSP-DMAE-NHS in anhydrous N, N-dimethylformamide, wherein the concentration of acridine ester is 5mg/mL;
preparation of acridine ester solution 5: dissolving an acridine salt NSP-DMAE-NHS in anhydrous N, N-dimethylformamide added with methylated-beta-cyclodextrin, wherein the concentration of acridine ester is 5mg/mL, and the molar concentration ratio of the methylated-beta-cyclodextrin to the acridine ester is 0.5:1;
preparation of acridine ester solution 6: dissolving an acridine salt NSP-DMAE-NHS in anhydrous N, N-dimethylformamide added with methylated-beta-cyclodextrin, wherein the concentration of acridine ester is 5mg/mL, and the molar concentration ratio of the methylated-beta-cyclodextrin to the acridine ester is 2:1;
preparation of acridine ester solution 7: dissolving the acridine salt NSP-SA-NHS in anhydrous dimethyl sulfoxide added with beta-cyclodextrin, wherein the concentration is 5mg/mL, and the molar concentration ratio of the beta-cyclodextrin to the acridine ester is 0.5:1;
preparation of acridine ester solution 8: dissolving the acridine salt NSP-SA-NHS in anhydrous dimethyl sulfoxide added with beta-cyclodextrin, wherein the concentration is 5mg/mL, and the molar concentration ratio of the beta-cyclodextrin to the acridine ester is 2:1;
subpackaging 1-6 acridine ester solution into 5 tubes respectively, and storing at-20 ℃ in a dark place; the tube 1 of each group was stored in-20 ℃ in the dark all the time after self-packaging, the tube 2 was taken out for re-melting and exposure for 5 minutes (simulated use scene), and then stored in-20 ℃ in the dark; the tube 3 is repeatedly taken out for re-melting and exposure for 5 minutes, and then is kept in the dark for 2 times at the temperature of minus 20 ℃; repeatedly taking out the tube 4 after the re-melting at room temperature, re-melting and exposing for 5 minutes, and then storing at-20 ℃ in a dark place for 3 times; repeatedly taking out the tube 5 after the re-melting at room temperature, re-melting and exposing for 5 minutes, and then storing at-20 ℃ in a dark place for 4 times; and finally, taking out all the tubes 1-5 of the acridine ester solutions 1-6 together for re-melting, respectively sucking 10uL of the acridine ester solutions, diluting the solution to 0.2ng/mL by using a labeling buffer solution, and detecting a luminescence signal value on a chemiluminescence immunoassay analyzer.
Other reagents required for detection comprise substrate liquid (comprising pre-excitation liquid and excitation liquid) and cleaning liquid, wherein the main component of the pre-excitation liquid is hydrogen peroxide, the main component of the excitation liquid is sodium hydroxide, acridinium ester and the substrate liquid are mixed to carry out chemical reaction, and an optical signal is released; the cleaning solution mainly contains a surfactant and is used for cleaning the pipeline of the instrument. The detection results of the luminous signals of the acridinium ester solution 1-6 after the simulated use process are shown in the following table 1:
TABLE 1 detection of luminescence signals after simulated application of acridinium ester solution 1-6
From the above data, it is understood that the optical signal attenuation amplitudes of the acridine ester solutions 1 and 4 are relatively large and decreased by 21.59% and 24.10% respectively after repeating the melting and exposure of the acridine ester solution 4 times, and that there is some signal attenuation in the acridine ester solutions 2, 3, 5, and 6 to which the methylated- β -cyclodextrin is added, but the attenuation amplitudes are 11.74% at the maximum, and the stability is superior to that of the acridine ester solution 1 and the acridine ester solution 4 to which the methylated- β -cyclodextrin is not added.
And (3) subpackaging the acridine ester solution 2-3,7-8 into 5 tubes, respectively sucking 10uL of the solution, diluting the solution to 0.2ng/mL by using a labeling buffer solution, and detecting a luminescence signal value on a chemiluminescence immunoassay analyzer.
TABLE 2. Detection results of luminescence signals after simulated use of acridinium ester solution 2-3,7-8
According to the data, detection signals between 2 tubes and 3 tubes of the acridinium ester solution are relatively uniform, and CV between the tubes is less than 2%; the difference of detection signals between 7 and 8 canals of the acridinium ester solution is large, and the CV between the canals is more than 20 percent;
example 3 comparison of the results of the luminous efficacy of acridinium ester-coupled immunoprotein complexes
The coupling of acridinium ester and an immunoprotein, which was PCT murine mAb (cat # 2A 7) from Wasabia chinensis (Kimura et al) and 25-hydroxy-vitamin D-bovine serum albumin conjugate from this company, respectively, was performed according to the procedure of example 1.
TABLE 3 preparation of acridinium ester-conjugated immunoprotein complexes
Purifying the solution 3-1 to 3-16 generated in the fourth step by a G50/G25 chromatographic column, diluting the solution to 0.01ug/mL by an acridinium ester storage buffer solution to generate acridinium ester coupled immune protein complexes 3-1 to 3-16, respectively detecting optical signals, and comparing detection results, wherein the detection results are shown in a table 4 and figures 2-4.
TABLE 4 luminous efficacy results for acridinium ester-coupled immunoprotein complexes
As can be seen from Table 4 and FIGS. 2-4, the comparison of the data of the experimental groups 3-1 to 3-5 shows that the luminous efficiency of the group 3-1 is the highest when a certain concentration of methylated-beta-cyclodextrin is added to the acridine ester solution as the control group; when the acridinium ester solution is added with the beta-cyclodextrin with a certain concentration, the luminous efficiency of the coupled antibody is even lower than that of a control group; the results of comparison of the data of the experimental groups 3-9 to 3-13 show that the luminous efficiency of the groups 3-9 is used as a control group, and when methylation-beta-cyclodextrin with a certain concentration is added into an acridine ester solution, the luminous efficiency of a coupled antigen is highest; when a certain concentration of beta-cyclodextrin is added to the acridinium ester solution, the luminous efficiency of the coupled antigen is even lower than or equal to that of the control group.
The acridine ester solution added with the methylated-beta-cyclodextrin and the acridine ester without the methylated-beta-cyclodextrin are respectively compared with the light-emitting signals after the coupling of the antibody and the antigen, and when the acridine ester solution contains the methylated-beta-cyclodextrin with a certain concentration in both the acridine ester coupled antibody compound and the acridine ester coupled antigen compound, the light-emitting signals after the coupling of the acridine ester and the immune protein are higher, which shows that the light-emitting efficiency is better.
While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A method of coupling an acridinium ester to an immunity protein, comprising: before the acridinium ester is coupled with the immune protein, the acridinium ester is dissolved in aprotic anhydrous N, N-dimethylformamide or anhydrous dimethyl sulfoxide added with methylated-beta-cyclodextrin, and then mixed.
2. The method of acridinium ester-coupled immunity protein of claim 1, wherein the concentration of the acridinium ester when mixed is 5mg/mL.
3. The method of acridinium ester-coupled immunity proteins of claim 2, wherein a molar concentration ratio of said methylated- β -cyclodextrin to said acridinium ester is 0.5:1-2:1.
4. the method of acridinium ester-coupled immunity proteins of claim 1, wherein said acridinium ester comprises acridinium salt NSP-DMAE-NHS or acridinium salt NSP-SA-NHS.
5. The method of acridinium ester-conjugated immunoprotein of claim 1, wherein the immunoprotein comprises an antibody and an antigen, optionally the antibody is murine immunoglobulin G and the antigen comprises 25-hydroxy-vitamin D-bovine serum albumin conjugate.
6. The method of acridinium ester-coupled immunoprotein of claim 5, wherein the concentration of the antibody is 1-2mg/mL; the concentration of the antigen is 0.5-1mg/mL.
7. The method for coupling acridinium esters to immunity proteins according to any one of claims 1 to 5, wherein the molar ratio of the acridinium ester to the immunity protein is: 10, 1 to 50.
8. The method of coupling acridinium esters with an immunity protein according to any one of claims 1 to 5, comprising the steps of:
step (1): dialyzing the immune protein;
step (2): absorbing the labeled buffer solution into a centrifuge tube to generate a solution 1;
and (3): sucking dialyzed immune protein into a centrifuge tube and uniformly mixing to generate a solution 2;
and (4): adding the acridine ester solution, uniformly mixing, and slowly oscillating at the temperature of 25 +/-2 ℃ for light-shielding reaction for 2 hours to generate a solution 3;
and (5): desalting and purifying the solution 3 to generate a solution 4;
and (6): adding an acridinium ester preservation buffer solution into the solution 4 to generate an acridinium ester coupled immune protein complex solution, and preserving at the temperature of 2-8 ℃ in a dark place.
9. The method for coupling acridinium ester with immune protein of claim 8, wherein the dialysis of step (1) uses a labeling buffer solution, wherein the labeling buffer solution is a phosphate buffer solution with pH value of 8.0 ± 0.2;
dissolving the acridine ester obtained in the step (4) in aprotic anhydrous N, N-dimethylformamide or anhydrous dimethyl sulfoxide added with methylated-beta-cyclodextrin, wherein the concentration of the acridine ester is 5mg/mL, and the molar concentration ratio of the methylated-beta-cyclodextrin to the acridine ester is 0.5:1-2:1; diluting the dissolved acridinium ester with the labeling buffer to a volume consistent with that of solution 2; the molar ratio of the acridinium ester to the immunoprotein is: 10, mixing the materials in parts by weight in a ratio of 1 to 50, and then slowly oscillating and keeping away from light at a temperature of 25 +/-2 ℃ for 2 hours to generate a solution 3;
the desalting purification of the step (5) is carried out by using a G50/G25 chromatographic column, and the excessive unbound acridinium ester, the methylated-beta-cyclodextrin and the acridinium ester-coupled immune protein complex are separated.
10. The application of the methylated-beta-cyclodextrin in preparing a reagent for improving the luminous signal and luminous efficiency of the immunoassay of the acridinium ester coupled immune protein.
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| CN115932284B (en) | 2023-07-21 |
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