CN114689835A - Method for coupling acridinium ester with antibody and application thereof - Google Patents
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- RXNXLAHQOVLMIE-UHFFFAOYSA-N phenyl 10-methylacridin-10-ium-9-carboxylate Chemical compound C12=CC=CC=C2[N+](C)=C2C=CC=CC2=C1C(=O)OC1=CC=CC=C1 RXNXLAHQOVLMIE-UHFFFAOYSA-N 0.000 title claims abstract description 55
- 238000000034 method Methods 0.000 title claims abstract description 36
- 238000010168 coupling process Methods 0.000 title claims abstract description 23
- 238000005859 coupling reaction Methods 0.000 title claims abstract description 23
- 230000008878 coupling Effects 0.000 title claims abstract description 22
- 238000003018 immunoassay Methods 0.000 claims abstract description 14
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 claims description 43
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims description 43
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 claims description 43
- 238000002156 mixing Methods 0.000 claims description 17
- 238000001514 detection method Methods 0.000 claims description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 10
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 2
- 229940098773 bovine serum albumin Drugs 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims 2
- 102000008394 Immunoglobulin Fragments Human genes 0.000 claims 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 claims 1
- 239000000243 solution Substances 0.000 description 18
- 238000006243 chemical reaction Methods 0.000 description 11
- 230000000052 comparative effect Effects 0.000 description 9
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 7
- 238000004020 luminiscence type Methods 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- -1 acridine ester Chemical class 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- XUVKSPPGPPFPQN-UHFFFAOYSA-N 10-Methyl-9(10H)-acridone Chemical compound C1=CC=C2N(C)C3=CC=CC=C3C(=O)C2=C1 XUVKSPPGPPFPQN-UHFFFAOYSA-N 0.000 description 4
- LXAHHHIGZXPRKQ-UHFFFAOYSA-N 5-fluoro-2-methylpyridine Chemical compound CC1=CC=C(F)C=N1 LXAHHHIGZXPRKQ-UHFFFAOYSA-N 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 238000011033 desalting Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000005081 chemiluminescent agent Substances 0.000 description 2
- 230000021615 conjugation Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 239000000891 luminescent agent Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 241001416177 Vicugna pacos Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000002038 chemiluminescence detection Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 230000005281 excited state Effects 0.000 description 1
- 230000005283 ground state Effects 0.000 description 1
- 230000036046 immunoreaction Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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Abstract
The invention discloses a method for coupling an acridinium ester with an antibody and application thereof, and relates to the technical field of immunoassay.
Description
Technical Field
The invention relates to the technical field of immunoassay, in particular to a method for preparing an acridinium ester coupling antibody and application thereof.
Background
Chemiluminescence immunoassay (CLIA) is a technique for detecting and analyzing various antigens or antibodies by combining a chemiluminescence assay technique with high sensitivity and a high specificity immunoreaction. Is a latest immunoassay technology developed after radioimmunoassay, enzyme immunoassay, fluoroimmunoassay and time-resolved fluoroimmunoassay.
Acridinium ester is a chemical substance which can be used as a chemiluminescent marker, and under alkaline conditions, the acridinium ester molecule is attacked by hydrogen peroxide to generate ethylene dioxide which is unstable and is decomposed into CO2And an electronically excited state of N-methylacridone which emits photons having a wavelength of 430nm when it returns to the ground state.
The acridinium ester marker has a special group generating luminescence on a chemical structure, and directly participates in a luminescence reaction in a luminescence immunoassay process. Generally, the substance has no background luminescence, can be used for detecting samples with low concentration or trace concentration in a reaction, and is a luminescent agent with high luminous efficiency. However, the acridinium ester labeled chemiluminescence detection often has false positive results, and the specificity of the detection needs to be improved.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method for preparing an acridinium ester coupling antibody and application thereof.
The invention is realized by the following steps:
in a first aspect, embodiments of the present invention provide a method for preparing an acridinium ester-conjugated antibody, comprising: the antibody was mixed with disodium EDTA and reacted prior to coupling the antibody to the acridinium ester.
In a second aspect, the embodiments of the present invention provide the use of disodium EDTA in the preparation of a reagent for improving the specificity of detection of acridinium ester labeled chemiluminescent immunoassays.
The invention can improve the reactivity of the coupling of the antibody and the acridine ester, the improvement is not limited to specific antibody types, and the antibody aiming at different antigens has effects. The most distinct regions of different antibodies are the CDRs, but the coupling to acridinium esters is at the Fc-terminus of the antibody. The inventors have validated the invention in a number of projects, the following of which is by way of illustration only.
The invention has the following beneficial effects:
the invention provides a method for coupling an acridinium ester antibody and application thereof, wherein EDTA disodium is firstly mixed with the antibody for reaction, and then the antibody after the mixed reaction is coupled with the acridinium ester.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and those skilled in the art can also obtain other related drawings based on the drawings without inventive efforts.
FIG. 1 shows the results of the linear detection of a calibrator in test example 1 of the present invention;
FIG. 2 shows the results of the linear detection of the calibrator in test example 2 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The features and properties of the present invention are described in further detail below with reference to examples.
Noun definitions
As used herein, "coupled" may refer to: the process of obtaining an organic molecule by a chemical reaction of two organic chemical units may also be referred to as conjugation.
As used herein, a "chemiluminescent immunoassay" is an immunoassay that is established by combining an antigen-antibody specific reaction with a sensitive chemiluminescent reaction. From the reaction principle, the chemiluminescence immunoassay technology is mainly divided into direct chemiluminescence and enzymatic reaction chemiluminescence. In direct chemiluminescence, chemiluminescent agents directly participate in luminescence reactions without the catalytic action of enzymes in the luminescence immunoassay process, and the chemiluminescent agents have specific groups generating luminescence on chemical structures and can directly mark antigens or antibodies. Representative luminescent agents include acridinium esters.
"acridinium esters" as described herein are tricyclic organic compounds, which are substituted by H under basic conditions2O2When oxidized, the compound emits light with the wavelength of 470nm and has high luminous efficiency, and the excited product N-methylacridone is the luminous body of the luminous reaction system.
The embodiment of the invention provides a method for preparing an acridinium ester coupled antibody, which comprises the following steps: the antibody was mixed with disodium EDTA and reacted prior to coupling the antibody to the acridinium ester.
The inventor discovers through a series of creative works that before the antibody and the acridinium ester are mixed and coupled, the EDTA disodium is adopted to pretreat the antibody, and the pretreated antibody and the acridinium ester conjugate are detected, so that the false positive phenomenon in chemiluminescence immunoassay can be effectively avoided or reduced, and the specificity of detection is effectively improved.
In some embodiments, the disodium EDTA is present in a working concentration of 1 to 10mM when mixed with the antibody. Specifically, the working concentration of disodium EDTA may be any of 1mM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, 4mM, 4.5mM, 5mM, 5.5mM, 6mM, 6.5mM, 7mM, 7.5mM, 8mM, 8.5mM, 9mM, 9.5mM, and 10 mM.
Preferably, the working concentration of disodium EDTA in admixture with the antibody is 2-5 mM. When disodium EDTA is within this concentration range, the specificity of detection is more effectively reduced or avoided.
Preferably, the mixing time of the disodium EDTA and the antibody is 5-40 min. In some embodiments, the mixing time of the disodium EDTA and the antibody may be any of 5min, 10min, 15min, 20min, 25min, 30min, 35min, and 40 min.
Preferably, the mixing temperature of the EDTA disodium and the antibody is 15-25 ℃. In some embodiments, the mixing temperature can be 15 degrees, 16 degrees, 17 degrees, 18 degrees, 19 degrees, 20 degrees, 21 degrees, 22 degrees, 23 degrees, 24 degrees, 25 degrees in any temperature.
Preferably, when the disodium EDTA is mixed with the antibody, the concentration of the antibody solution is 1-3 mg/mL. In some embodiments, the concentration of the antibody solution can be any of 1mg/mL, 1.5mg/mL, 2mg/mL, 2.5mg/mL, 3 mg/mL.
In some embodiments, the acridinium ester is mixed with the antibody solution in the following manner: after mixing, the mixture was reacted at room temperature in the dark. The method specifically comprises the following steps: immediately mixing the mixture in a small vortex oscillator by vortex oscillation for 10 s; and after the centrifugal separation in a small centrifugal machine, reacting for 40-80 minutes at room temperature in a dark place, and then sealing by adopting a glycine solution.
In some embodiments, after the acridinium ester is mixed with the antibody for conjugation, the method further comprises subjecting the mixture or conjugate to a purification step.
Preferably, the purification step comprises: the coupled conjugate was added to a 0.5ml 7k desalting column, centrifuged at 1500 xg for 2min in a high speed centrifuge, the desalted marker was collected, the desalted and purified conjugate was adjusted to a certain concentration using PBS (20mM PB and 150mM NaCl, pH 7.4), added to an equal volume of high pressure glycerol and mixed well, and stored at-20 ℃.
The antibody of the present invention is not particularly limited and may be selected from any antibody type that can be labeled with acridinium ester. Can be one or two of genetically engineered antibody, polyclonal antibody and monoclonal antibody. The antibody can be one or more of murine antibody, rabbit antibody, sheep antibody and alpaca antibody according to source classification.
When the antibody is coupled with the acridine ester, the concentration of the acridine ester solution is 0.05-2 mM, specifically any concentration of 0.05mM, 0.10mM, 0.15mM and 0.2 mM; the amount of the antibody to be added is 50 to 1000. mu.g, and specifically may be any of 50. mu.g, 100. mu.g, 150. mu.g, 200. mu.g, 250. mu.g, 300. mu.g, 400. mu.g, 500. mu.g, 600. mu.g, 700. mu.g, 800. mu.g, 900. mu.g, and 1000. mu.g.
Preferably, the conditions for coupling the antibody to the acridinium ester are: and mixing the mixture at 15-25 ℃ in the dark for 10-60 min, adding bovine serum albumin BSA solution, and mixing the mixture in the dark for 10-60 min. The light-shielding mixing is preferably performed by shaking, and the time can be any of 10min, 20min, 30min, 40min, 50min and 60 min.
Preferably, when the antibody is coupled with the acridinium ester, the coupling buffer solution adopted is carbonate CBS buffer solution;
preferably, the CBS buffer comprises: 15 to 20mM CB and 130 to 170mM NaCl, pH 9 to 10.
In addition, the embodiment of the invention also provides application of the EDTA disodium in preparing a reagent for improving the detection specificity of the acridinium ester labeled chemiluminescence immunoassay.
Unless otherwise indicated, all chemical reagents used in the examples of the present invention were analytical grade and were obtained from Sigma-aldrich. The chemiluminescence detector is a Bincho BHP9507 chemiluminescence detector of Beijing Bincho photon technology corporation in China.
Example 1
A method of acridinium ester-conjugated antibodies comprising the steps of:
(1) antibody treatment:
taking a certain amount of antibody to be marked, desalting, replacing and purifying the antibody to be marked to carbonate CBS (20mM CB and 150mM NaCl pH 9.5) buffer solution, averagely dividing the antibody after replacement and purification into two parts, taking one part of the antibody solution, adding disodium EDTA, using the CBS solution to set the concentration of the antibody to be 2mg/mL, setting the working concentration of the disodium EDTA to be 5mM, and reacting the antibody and the disodium EDTA at room temperature for half an hour after fully and uniformly mixing; another antibody solution was directly used as CBS solution to the same concentration of 2mg/ml for use.
(2) Coupling of the antibody with acridinium ester:
2.1, measuring 12 mM CBS (20mM CB and 150mM NaCl pH 9.5) buffer solution in a centrifuge tube;
2.2, adding 1mg of the antibody to be marked, and fully and uniformly mixing;
2.3, adding 400 mu L of acridine ester solution (1mg/mL), fully and uniformly mixing, and shaking for 1 hour at room temperature in a dark place;
2.4, adding 1.4mL of 10% BSA solution, mixing uniformly, and carrying out oscillation reaction for 30 minutes at room temperature in a dark place;
2.5, the product was put into a 30mL G25 desalting column, centrifuged at 1500 xg for 2min in a high speed centrifuge, the desalted marker was collected, the desalted and purified conjugate was adjusted to a certain concentration using PBS (20mM PB and 150mM NaCl, pH 7.4), and the mixture was mixed with an equal volume of glycerol and stored at-20 ℃.
Example 2
A method of acridinium ester-conjugated antibodies, substantially as in example 1, except that the working concentration of disodium EDTA is varied as follows:
the working concentration of disodium EDTA was 4 mM.
Example 3
A method of acridinium ester-conjugated antibodies, substantially as in example 1, except that the working concentration of disodium EDTA is varied as follows:
the working concentration of disodium EDTA was 3 mM.
Example 4
A method of acridinium ester-conjugated antibodies, substantially as in example 1, except that the working concentration of disodium EDTA is varied as follows:
the working concentration of disodium EDTA was 2 mM.
Example 5
A method of acridinium ester-conjugated antibodies, substantially as in example 1, except that the working concentration of disodium EDTA is varied as follows:
the working concentration of disodium EDTA was 1 mM.
Example 6
A method of coupling acridinium esters to antibodies, substantially as in example 1, except that the working concentration of disodium EDTA is different as follows:
the working concentration of disodium EDTA was 8 mM.
Example 7
A method of acridinium ester-conjugated antibodies, substantially as in example 1, except that the working concentration of disodium EDTA is varied as follows:
the working concentration of disodium EDTA was 10 mM.
Comparative example 1
A method of acridinium ester-conjugated antibodies, substantially the same as example 1, except that disodium EDTA is not added.
Comparative example 2
A method of coupling acridinium ester to an antibody, substantially as in example 1, except that the timing of the disodium EDTA addition was different, in this comparative example the disodium EDTA addition was: after the acridinium ester is mixed with the antibody, it is added as a buffer.
In comparative example 1, the working concentration of disodium EDTA was 5 mM.
Test example 1
Experimental methods
CA153 antibody (antibody to be labeled) and acridinium ester solution are coupled by the method of acridinium ester coupling antibody provided in example 1 and comparative example 1 respectively, and the coupled antibody is used for detecting a calibrator and a clinical sample.
The test results of the calibrator are shown in table 1.
TABLE 1 calibrator test results
As can be seen from Table 1 and FIG. 1, the better linearity value R can be obtained by pretreating the antibody with disodium EDTA20.99, and the linear range of the calibrator can reach 1000U/mL.
The results of the clinical sample tests are shown in Table 2.
TABLE 2 clinical specimen test results
| Clinical samples (U/mL) | Example 1(U/mL) | COMPARATIVE EXAMPLE 1(U/mL) |
| Sample 1: 19.6 | 20.118 | 100.1669 |
| Sample 2: 22.7 | 21.2277 | 40.0903 |
| Sample 3: 24.8 | 23.1523 | 28.6699 |
| Sample 4: 26.8 | 27.1016 | 28.6209 |
| Sample 5: 85.4 | 84.4134 | 79.5694 |
| Sample 6: 137.9 | 132.0966 | 153.8142 |
As can be seen from table 2, the antibody is pretreated with disodium EDTA, which can improve the accuracy of detection and the influence of the antiserum matrix. Comparative example 1 false positive results were detected in both clinical samples 1 and 2, and the values detected using the method of the invention corresponded to the clinical values.
Test example 2
Experimental methods
The acridinium ester conjugated antibody provided in example 1 and comparative example 2 was used to conjugate CA153 antibody and acridinium ester solution, and the conjugated antibody was used to detect calibrator and clinical specimen.
The test results of the calibrator are shown in Table 3.
TABLE 3 test results of calibrators
As can be seen from Table 3 and FIG. 2, the better linearity R can be obtained by pre-treating the antibody with disodium EDTA20.99, and the linear range of the calibrator can reach 1000U/mL. The addition of disodium EDTA to the labeled dilutions did not improve the linearity and detection range of the assay.
The results of the clinical samples are shown in Table 4.
TABLE 4 clinical specimen test results
| Clinical samples (U/mL) | Example 1(U/mL) | COMPARATIVE EXAMPLE 1(U/mL) |
| Sample 1: 19.6 | 20.118 | 130.1576 |
| Sample 2: 22.7 | 21.2277 | 58.1648 |
From the results, it was found that the addition of disodium EDTA after the labeling could not improve the detection of the false positive sample of test example 1.
Test example 3
Experimental methods
The method of acridinium ester coupling antibody provided by the embodiments 1-7 is respectively adopted to couple CA153 antibody and acridinium ester solution, and the coupled antibody is adopted to detect the calibrator and clinical samples.
The test results are shown in Table 5.
TABLE 5 test results
| Sample 1(U/mL) | Sample 2(U/mL) | |
| Example 1 | 20.118 | 21.2277 |
| Example 2 | 19.684 | 22.613 |
| Example 3 | 20.038 | 22.164 |
| Example 4 | 18.217 | 22.717 |
| Example 5 | 17.846 | 20.163 |
| Example 6 | 21.012 | 24.384 |
| Example 7 | 21.274 | 24.891 |
From the results, in examples 1-7, after the CA153 antibody is pretreated with disodium EDTA at different concentrations of 1-10mM, the antibody is coupled with acridinium ester for labeling, and the detection is carried out by using the clinical samples 1 and 2 which are easy to detect as false positives, and after the pretreatment with disodium EDTA at 1-10mM before the antibody coupling labeling, the detection value is consistent with the clinical actual value, and no false positive result is shown. The particular examples of 2-5mM disodium EDTA pre-treatment deviate by no more than 5% from clinical values.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method of coupling an acridinium ester to an antibody, comprising: the antibody was mixed with disodium EDTA and reacted prior to coupling the antibody to the acridinium ester.
2. The method for preparing an acridinium ester-conjugated antibody according to claim 1, wherein the working concentration of disodium EDTA in the mixture with the antibody is 1-10 mM.
3. The method of acridinium ester-conjugated antibodies according to claim 2, wherein the working concentration of disodium EDTA in the antibody mixture is 2-5 mM.
4. The method of acridinium ester-conjugated antibodies of claim 1, wherein the mixing reaction time of disodium EDTA and antibody is at least 5 min;
the mixing reaction time is preferably 5-40 min.
5. The method for preparing an acridinium ester-conjugated antibody according to claim 1, wherein when disodium EDTA is mixed with the antibody, the concentration of the antibody solution is 1-3 mg/mL.
6. The method for preparing an acridinium ester-conjugated antibody according to claim 5, wherein the antibody is any one or more of a genetically engineered antibody, a monoclonal antibody, a polyclonal antibody or an antibody fragment.
7. The method for preparing an acridinium ester-coupled antibody according to any one of claims 1 to 5, wherein the concentration of the acridinium ester solution is 0.05 to 2mM and the amount of the antibody added is 50 to 1000. mu.g when the antibody is coupled to an acridinium ester.
8. The method of acridinium ester-conjugated antibodies of claim 7, wherein said antibody-coupling conditions are: and mixing the mixture at 15-25 ℃ in a dark place for 10-60 min, adding a bovine serum albumin BSA solution, and mixing the mixture in a dark place for 10-60 min.
9. The method for coupling the acridinium ester with the antibody according to claim 7, wherein when the antibody is coupled with the acridinium ester, a coupling buffer adopted is a carbonate CBS buffer;
preferably, the CBS buffer comprises: 15 to 20mM CB and 130 to 170mM NaCl, pH 9 to 10.
Use of disodium EDTA in the preparation of a reagent for improving the detection specificity of an acridinium ester labeled chemiluminescent immunoassay.
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| CN115932284A (en) * | 2023-03-10 | 2023-04-07 | 普迈德（北京）科技有限公司 | Method for coupling acridine ester with immune protein and application thereof |
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| CN111175496A (en) * | 2020-02-27 | 2020-05-19 | 江苏泽成生物技术有限公司 | Pregnancy-associated protein A magnetic particle chemiluminescence detection kit and use method thereof |
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