CN113533735A - Application of acridinium ester analogue in preparation of acridinium ester analogue labeled antibody - Google Patents

Application of acridinium ester analogue in preparation of acridinium ester analogue labeled antibody Download PDF

Info

Publication number
CN113533735A
CN113533735A CN202110783301.6A CN202110783301A CN113533735A CN 113533735 A CN113533735 A CN 113533735A CN 202110783301 A CN202110783301 A CN 202110783301A CN 113533735 A CN113533735 A CN 113533735A
Authority
CN
China
Prior art keywords
acridinium ester
ester analog
antibody
labeled
antigen
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
CN202110783301.6A
Other languages
Chinese (zh)
Inventor
曹亦菲
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Boyixin Hangzhou Biotechnology Co ltd
Original Assignee
Boyixin Hangzhou Biotechnology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Boyixin Hangzhou Biotechnology Co ltd filed Critical Boyixin Hangzhou Biotechnology Co ltd
Priority to CN202110783301.6A priority Critical patent/CN113533735A/en
Publication of CN113533735A publication Critical patent/CN113533735A/en
Withdrawn legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
    • G—PHYSICS
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
    • G—PHYSICS
    • 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
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Endocrinology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention provides an application of an acridinium ester analog in preparation of an acridinium ester analog labeled antibody, belonging to the technical field of biomedical detection. In the invention, the acridinium ester analog is used for labeling the antibody, and the antigen in the sample and the enzyme-labeled antigen compete to be combined with the acridinium ester analog labeled antibody to form an immune complex. The acridine ester analog and the enzyme in the compound are close to each other, the luminous substrate acridine ester analog is catalyzed and cracked by the enzyme in the compound under the action of the excitation liquid to form an unstable excited state intermediate, when the excited state intermediate returns to the ground state, photons are emitted, the number of the generated photons is inversely proportional to the concentration of the antigen in the sample, and accordingly the concentration of the antigen in the sample can be calculated. The acridinium ester analogue is used for competitive detection, the detection antibody can perform immunoreaction with an analyte to be detected in a homogeneous system, a microporous plate, magnetic beads or microspheres are not required to serve as a carrier coating antibody, and the separation-free chemiluminescence technology is provided.

Description

Application of acridinium ester analogue in preparation of acridinium ester analogue labeled antibody
Technical Field
The invention relates to the technical field of biomedical detection, in particular to application of an acridinium ester analog in preparation of an acridinium ester analog labeled antibody.
Background
Immunodiagnosis (immunodiagnosis) is the application of immunological theories, techniques and methods to diagnose a variety of diseases and determine immune status. The immunodetection reagent has the most varieties in diagnostic kits, is widely applied to hospitals, blood stations and physical examination centers, and is mainly used for hepatitis detection, venereal disease detection, tumor detection, pregnancy detection and the like. Methods for immunoassay include sandwich, indirect and competitive methods. The current immunoassay is mainly a competition method, and the basic principle of the competition method is as follows: the antigen or antibody is combined to the surface of some solid phase carrier and its immunological activity is maintained. Secondly, the antigen or antibody is connected with a certain enzyme to form an enzyme-labeled antigen or enzyme-labeled antibody, and the enzyme-labeled antigen or enzyme-labeled antibody not only retains the immunological activity of the antigen or antibody, but also retains the activity of the enzyme.
In the measurement, the specimen to be tested (the antibody or antigen to be measured therein) and the enzyme-labeled antigen or antibody are reacted with the antigen or antibody on the surface of the solid carrier in a different step. The antigen-antibody complex formed on the solid phase carrier is separated from other substances by washing, and finally the enzyme quantity bound on the solid phase carrier is in a certain proportion to the quantity of the detected substance in the specimen. After the substrate of the enzyme reaction is added, the substrate is catalyzed by the enzyme to be changed into a colored product, and the amount of the product is directly related to the amount of the detected substance in the sample, so that qualitative or quantitative analysis can be carried out according to the shade of the color reaction. However, the antigen or antibody needs to be bound to a solid phase carrier, and the operation is complicated.
Disclosure of Invention
The invention aims to provide application of acridinium ester analogs in preparation of acridinium ester analog labeled antibodies, and the acridinium ester analogs are used for detecting the acridinium ester analog labeled antibodies by a competition method, so that the limitation of solid phase carriers can be eliminated.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides application of acridinium ester analogues in preparation of acridinium ester analogue labeled antibodies;
the molecular structural formula of the acridine ester analog is shown as a formula I;
Figure BDA0003158053490000021
the invention also provides the application of the acridinium ester analogue in preparing a competitive method detection kit; the molecular structural formula of the acridine ester analog is shown as a formula I;
Figure BDA0003158053490000022
the invention also provides a competitive method detection kit, which comprises an acridinium ester analogue labeled antibody, an enzyme labeled antigen and an excitation liquid;
the molecular structural formula of the acridine ester analog is shown as a formula I;
Figure BDA0003158053490000023
preferably, a background inhibitor is also included.
Preferably, the background inhibitor comprises a hydroxy diethylamine aqueous solution with the molar concentration of 20-30 mM.
Preferably, the exciting liquid comprises the following components in concentration: 20 to 30mM tris, 0.5 to 1.5mM p-phenylphenol, 0.3 to 0.8mM EDTA, 0.1 to 0.3% Tween-20 and 3 to 8mM carbamide peroxide; the pH value of the tris is 7.8-8.2.
Preferably, the enzyme-labeled antigen comprises a horseradish peroxidase-labeled antigen.
Preferably, the competitive method detection kit comprises a T3 detection kit, a T4 detection kit, a clenbuterol detection kit or a ractopamine detection kit.
Preferably, the acridinium ester analog labeled antibody comprises an acridinium ester analog labeled T3 monoclonal antibody, an acridinium ester analog labeled T4 monoclonal antibody, an acridinium ester analog labeled clenbuterol monoclonal antibody, or an acridinium ester analog labeled ractopamine monoclonal antibody.
The invention also provides a competitive method detection method for non-diagnosis purposes, which comprises the following steps:
1) labeling the antibody with an acridinium ester analog;
2) mixing the antibody marked with the acridine ester analog, the enzyme-labeled antigen and a sample to be detected, and incubating at 35-40 ℃ for 12-15 min to obtain a reaction solution;
3) adding an excitation liquid into the reaction liquid, detecting a luminescent signal generated within 1 second after the excitation liquid is added, and obtaining the antigen concentration in the sample to be detected according to a standard curve and the luminescent signal;
the standard curve is obtained by establishing a luminous signal value obtained by a calibration object with gradient concentration and a concentration value corresponding to the calibration object;
the molecular structural formula of the acridine ester analog is shown as a formula I;
Figure BDA0003158053490000031
the invention has the beneficial effects that: the invention provides application of acridinium ester analogues in preparation of acridinium ester analogue labeled antibodies; the molecular structural formula of the acridine ester analog is shown as a formula I;
Figure BDA0003158053490000032
in the invention, the acridinium ester analog is used for labeling the antibody, and the antigen in the sample and the enzyme-labeled antigen compete to be combined with the acridinium ester analog labeled antibody to form an immune complex. The acridine ester analogue and the enzyme in the immune complex are close to each other, the luminous substrate acridine ester analogue is catalyzed and cracked by the enzyme in the complex under the action of the excitation liquid to form an unstable excited state intermediate, when the excited state intermediate returns to the ground state, photons are emitted, the number of the generated photons is inversely proportional to the concentration of the antigen in the sample, and accordingly the concentration of the antigen in the sample can be calculated. The acridinium ester analogue is used for competitive detection, the detection antibody can perform immunoreaction with an analyte to be detected in a homogeneous system, a microporous plate, magnetic beads or microspheres are not required to serve as a carrier coating antibody, and the separation-free chemiluminescence technology is provided. The method is simple and efficient to operate, only needs about 15min for detection, and greatly shortens the detection time. The acridinium ester analogue can be used for homogeneous phase, rapid and high-sensitivity competitive method detection, and is simpler and more efficient compared with the known competitive method. In addition, the method has the advantages of small antibody dosage, low cost, simple production process, easy amplification production, convenient detection process, low requirement on detection instruments and easy realization of full automation.
Drawings
FIG. 1 is a schematic diagram of competitive assay T3/T4;
FIG. 2 is a standard curve of competitive assay for T3, wherein T3 is diluted in PBS buffer and serum, respectively;
FIG. 3 is a standard curve of competitive assay for T4, wherein T3 is diluted in PBS buffer and serum, respectively;
FIG. 4 is a standard curve for detecting CLE by the immuno-competitive method, wherein CLE is diluted in PBS buffer and urine, respectively;
FIG. 5 is a standard curve for the detection of RAC by the immuno-competitive assay, wherein RAC is diluted in PBS buffer and urine, respectively.
Detailed Description
The invention provides application of acridinium ester analogues in preparation of acridinium ester analogue labeled antibodies;
the molecular structural formula of the acridine ester analog is shown as a formula I;
Figure BDA0003158053490000041
the invention also provides the application of the acridinium ester analogue in preparing a competitive method detection kit; the molecular structural formula of the acridine ester analog is shown as a formula I;
Figure BDA0003158053490000051
in the invention, the acridinium ester analog labeled antibody is preferably an acridinium ester analog labeled antibody in a competitive method detection kit.
The invention also provides a competitive method detection kit, which comprises an acridinium ester analogue labeled antibody, an enzyme labeled antigen and an excitation liquid;
the molecular structural formula of the acridine ester analog is shown as a formula I;
Figure BDA0003158053490000052
in the invention, the acridinium ester analogues have light energy transfer activity and are used for labeling antibodies and detecting luminescence.
In the present invention, the acridinium ester analogs are conventionally commercially available. In the specific implementation process of the invention, the acridinium ester analog is purchased from Lumigen corporation in America.
In the present invention, the excitation liquid preferably comprises the following components in concentrations: 20 to 30mM tris, 0.5 to 1.5mM p-phenylphenol, 0.3 to 0.8mM EDTA, 0.1 to 0.3% Tween-20 and 3 to 8mM carbamide peroxide; the pH value of the tris is 7.8-8.2; more preferably, the exciting liquid preferably comprises the following components in concentration: 22 to 28mM tris, 0.8 to 1.2mM p-phenylphenol, 0.4 to 0.6mM EDTA, 0.15 to 0.25% Tween-20 and 4 to 6mM carbamide peroxide; the pH value of the tris is 7.8-8.2; most preferably, the exciting liquid comprises the following components in concentration: 25mM tris, 1mM p-phenylphenol, 0.5mM EDTA, 0.2% Tween-20 and 5mM carbamide peroxide; the pH of the tris was 8.0.
In the invention, the exciting liquid provides a reactant, and can generate chemiluminescence reaction with the excited intermediate compound.
In the invention, the acridinium ester analog is used for labeling the antibody, and the antigen in the sample and the enzyme-labeled antigen compete to be combined with the acridinium ester analog labeled antibody to form an immune complex. The acridine ester analogue and the enzyme in the compound are close to each other, the luminescent substrate is catalyzed and cracked by the enzyme in the compound under the action of the excitation liquid to form an unstable excited state intermediate, when the excited state intermediate returns to the ground state, photons are emitted, the number of the generated photons is inversely proportional to the concentration of the antigen in the sample, and accordingly the concentration of the antigen in the sample can be calculated.
In the invention, the excitation liquid is used for exciting a luminescent signal, so that the detection sensitivity and the repeatability are improved.
In the present invention, the competitive assay kit preferably further comprises a background inhibitor; the background inhibitor comprises a hydroxy diethylamine aqueous solution with the molar concentration of 20-30 mM.
In the present invention, the molar concentration of the aqueous solution of hydroxydiethylamine is preferably 25 mM.
In the invention, the volume ratio of the acridinium ester analogue labeled antibody, the enzyme labeled antigen, the exciting liquid and the background inhibitor is preferably 10: 10: 10: (0.5-2), preferably 10: 10: 10: 1.
the invention applies the acridinium ester analog with light energy transfer, eliminates interference by adding a background inhibitor, and reduces the background signal value. In the invention, the unbound excessive antibody, acridinium ester analogue and catalase are not required to be washed and removed, and the chemiluminescence reaction and the detection result are not influenced under the action of a background inhibitor.
In the present invention, the enzyme-labeled antigen preferably includes an antigen labeled with horseradish peroxidase. In the present invention, the antigen is preferably T3, T4, clenbuterol small molecule marker BSA carrier protein (CLE-BSA) or ractopamine small molecule marker BSA carrier protein (RAC-BSA); the T3, T4, CLE-BSA and RAC-BSA were from conventional commercial sources, and in the practice of the present invention, the T3 or T4 were from Sigma, USA or from Aladdin Biotechnology GmbH. In the present invention, the enzyme-labeled antigen is preferably obtained by a conventional preparation method.
In the present invention, the competitive assay kit preferably includes a T3 assay kit, a T4 assay kit, a clenbuterol assay kit or a ractopamine assay kit.
In the present invention, the acridinium ester analog-labeled antibody preferably comprises an acridinium ester analog-labeled T3 monoclonal antibody, an acridinium ester analog-labeled T4 monoclonal antibody, an acridinium ester analog-labeled clenbuterol monoclonal antibody or an acridinium ester analog-labeled ractopamine monoclonal antibody.
The invention also provides a competitive method detection method for non-diagnosis purposes, which comprises the following steps:
1) labeling the antibody with an acridinium ester analog;
2) mixing the antibody marked with the acridine ester analog, the enzyme-labeled antigen and a sample to be detected, and incubating for 12-15 min at 35-40 ℃ to obtain a reaction solution;
3) adding an excitation liquid into the reaction liquid, detecting a luminescent signal generated within 1 second after the excitation liquid is added, and calculating the concentration of the antigen in the sample according to a standard curve; the standard curve is established by a luminous signal value obtained by a calibration object with gradient concentration and a concentration value corresponding to the luminous signal value.
The invention firstly adopts acridine ester analog to mark antibody; the molecular structural formula of the acridine ester analog is shown as a formula I;
Figure BDA0003158053490000071
in the present invention, when the antibody is a monoclonal antibody, the monoclonal antibody is a T3 monoclonal antibody or a T4 monoclonal antibody, the method for preparing the acridinium ester analog-labeled antibody comprises the following steps:
s1, mixing the acridine ester analog solution, the monoclonal antibody solution and a boric acid buffer solution, and carrying out coupling reaction to obtain a coupling reaction solution;
s2, dialyzing the coupling reaction solution, and collecting a part with the cut-off molecular weight of more than 7000 to obtain the acridinium ester analogue labeled antibody.
In the present invention, the solvent of the acridinium ester analog solution preferably comprises N, N-dimethylformamide. In the invention, the mass volume concentration of the acridine ester analogue solution is preferably 0.3-0.8 mg/mL, and more preferably 0.5 mg/mL; the mass volume concentration of the monoclonal antibody solution is preferably 8-12 mg/mL, and more preferably 10 mg/mL; the molar concentration of the boric acid buffer solution is preferably 0.03-0.08M, and more preferably 0.05M; the volume ratio of the acridine ester analog solution to the monoclonal antibody solution to the boric acid buffer solution is preferably (0.5-1.5): (0.5-1.5): (6-10), more preferably 1: 1: 8.
in the invention, the temperature of the coupling reaction is preferably 20-30 ℃, and more preferably 25 ℃; the time of the coupling reaction is preferably 0.8-1.2 h, and more preferably 1 h; the coupling reaction is preferably carried out under the condition of rotation, and the rotation speed of the rotation is preferably 20-60 r/min, and more preferably 40 r/min.
In the present invention, the dialysis process preferably includes: adding the coupling reaction solution into a dialysis bag with the molecular weight cutoff of 7000, and dialyzing in a PBS buffer solution at 4 ℃ for 20-30 h; replacing the PBS buffer solution for 2-3 times by the breathable flagship to obtain a T3 monoclonal antibody (T3-A) marked with the acridine ester analog or a T4 monoclonal antibody (T4-A) marked with the acridine ester analog; the dialysis time is preferably 24 h; the T3 monoclonal antibody (T3-A) marked with the acridine ester analog or the T4 monoclonal antibody (T4-A) marked with the acridine ester analog is preserved in a preservation solution; the preservation temperature is preferably-20 ℃; the preservation solution is preferably added with 0.05% of Proclin-300 by volume concentration on the basis of PBS buffer solution.
After the acridine ester analog is adopted to mark the antibody, the antibody marked with the acridine ester analog, the enzyme-labeled antigen and a sample to be detected are mixed, and incubated for 12-15 min at 35-40 ℃ to obtain a reaction solution.
In the present invention, the temperature of the incubation is preferably 37 ℃; the incubation time is preferably 13 min.
After the reaction liquid is obtained, adding an excitation liquid into the reaction liquid, detecting a luminescent signal generated within 1 second after the excitation liquid is added, and obtaining the antigen concentration in a sample to be detected according to a standard curve and the luminescent signal; the standard curve is established by a luminous signal value obtained by a calibration object with gradient concentration and a concentration value corresponding to the luminous signal value.
In the present invention, the calculation formula of detection T3 is: y 2.64594-0.59938x (r)20.95552); the calculation formula for test T4 is: 3.34862-0.49008x (r 2 0.92875), wherein yRepresents the base 10 logarithm of the luminescence signal value, and x represents the base 10 logarithm of the concentration of the standard.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
A schematic representation of the competitive assay for detection of T3/T4 is shown in FIG. 1.
The molecular structural formula of the acridine ester analogue is shown in formula I, and the acridine ester analogue is purchased from Lumigen corporation in America;
Figure BDA0003158053490000091
example 1
Horseradish peroxidase was conjugated to triiodothyronine (T3) and the T3 monoclonal antibody was conjugated to acridinium ester analogues for detection by an immuno-competitive assay. The following steps are carried out in sequence:
1. horseradish peroxidase conjugated T3
Mu.g of triiodothyronine derivative (T3-NHS) was added to 1ml of 10mM carbonate buffer (pH8.0), 100. mu.g of horseradish peroxidase was added thereto, and the mixture was stirred gently at 37 ℃ for 2 hours in the absence of light. Purifying the reaction solution by adding SUPERDEX200 gel filtration column, collecting the coupling product (HRP-T3) of purified HRP and T3 polymerase, and storing at-20 deg.C with a storage solution (PBS + 0.05% Proclin-300).
Coupling of acridinium ester analogs to T3 monoclonal antibodies
1mg of the luminescent substrate acridinium ester analog (A) is dissolved in 2ml of DMF, 100. mu.l of the dissolved acridinium ester analog is added to 100. mu.l of T3 monoclonal antibody with the concentration of 10mg/ml, and then 800. mu.l of 0.05M boric acid buffer is added, and the reaction is rotated at room temperature for 1 h. 1ml of the reaction solution was put into a dialysis bag with a molecular weight cutoff of 7000, and dialyzed in PBS buffer at 4 ℃ for 1 day, during which time the PBS buffer was changed 3 times, to obtain T3 monoclonal antibody (T3-A) linked to the acridine ester analog, and finally stored at-20 ℃ with 1ml of a storage solution (PBS + 0.05% Proclin-300).
3. Detection of
Preparing an exciting liquid: 25mM tris (pH8.0), 1mM p-phenylphenol, 0.5mM EDTA, 0.2% Tween-20 and 5mM carbamide peroxide.
Preparing a background inhibitor: 25mM hydroxydiethylamine in deionized water.
T3 sample: t3 standard samples were diluted with PBS buffer (pH 7.4) to different concentrations of 100ng/ml, 10ng/ml, 1ng/ml, 0.1ng/ml, 0.01ng/ml and stored at 4 ℃ as calibrators. Another T3 standard was diluted to 100ng/ml, 10ng/ml, 1ng/ml, 0.1ng/ml, 0.01ng/ml with T3 negative serum.
Sample adding detection: adopting a competition method, diluting the prepared HRP-T3 and T3-A by 15000 times and 2000 times respectively by PBS buffer solution, adding 100 mu l of the sample to be detected and 10 mu l of background inhibitor into a transparent reaction tube respectively during detection by HRP-T3 and T3-A. After incubation for 15min at 37 ℃ the assay was started immediately by adding 100. mu.l of excitation solution.
The calculation formula for test T3 is: y 2.64594-0.59938x (r)20.95552) where y represents the base 10 logarithm of the luminescence signal value and x represents the base 10 logarithm of the concentration of the standard.
And (3) detection results:
as shown in FIG. 2, T3 was detected by the acridinium ester analog separation-free chemiluminescence immune competition method, and the minimum detection limit of T3 of PBS gradient dilution was 0.01ng/ml, which is substantially the same as the detection sensitivity in serum. The linear range is 3 orders of magnitude.
Example 2
Horseradish peroxidase was conjugated to thyroxine (T4) and the T4 monoclonal antibody was conjugated to acridinium ester analogs for detection by an immuno-competitive assay. The following steps are carried out in sequence:
1. horseradish peroxidase conjugated T4
Mu.g of thyroxine derivative (T4-NHS) was added to 1ml of 10mM carbonate buffer (pH8.0), 100. mu.g of horseradish peroxidase was added thereto, and the mixture was stirred gently at 37 ℃ for 2 hours in the absence of light. Purifying the reaction solution by adding SUPERDEX200 gel filtration column, collecting the coupling product (HRP-T4) of purified HRP and T4 polymerase, and storing at-20 deg.C with a storage solution (PBS + 0.05% Proclin-300).
Coupling of acridinium ester analogs to T4 monoclonal antibodies
1mg of the luminescent substrate acridinium ester analog (A) is dissolved in 2ml of DMF, 100. mu.l of the dissolved acridinium ester analog is added to 100. mu.l of T4 monoclonal antibody with the concentration of 10mg/ml, and then 800. mu.l of 0.05M boric acid buffer is added, and the reaction is rotated at room temperature for 1 h. 1ml of the reaction solution was put into a dialysis bag with a molecular weight cutoff of 7000, and dialyzed in PBS buffer at 4 ℃ for 1 day, during which time the PBS buffer was changed 3 times, to obtain T3 monoclonal antibody (T3-A) linked to the acridine ester analog, and finally stored at-20 ℃ with 1ml of a storage solution (PBS + 0.05% Proclin-300).
3. Detection of
Preparing an exciting liquid: 25mM tris (pH8.0), 1mM p-phenylphenol, 0.5mM EDTA, 0.2% Tween-20 and 5mM carbamide peroxide.
Preparing a background inhibitor: 25mM hydroxydiethylamine in deionized water.
T4 sample: t4 standard samples were diluted with PBS buffer (pH 7.4) to different concentrations of 1000ng/ml, 100ng/ml, 10ng/ml, 1ng/ml, 0.1ng/ml, and stored at 4 ℃ as calibrators. Another T4 standard was diluted to 1000ng/ml, 100ng/ml, 10ng/ml, 1ng/ml, 0.1ng/ml with T4 negative serum.
Sample adding detection: adopting a competition method, respectively diluting the HRP-T4 and the T4-A prepared by the method by 20000 times and 4000 times by using PBS buffer, respectively adding 100 mu l of the HRP-T4 and the T4-A to the sample to be detected during detection, and additionally adding 10 mu l of background inhibitor into a transparent reaction tube. After incubation for 15min at 37 ℃ the assay was started immediately by adding 100. mu.l of excitation solution.
The calculation formula for test T4 is: y 3.34862-0.49008x (r)20.92875) where y represents the base 10 logarithm of the luminescence signal value and x represents the base 10 logarithm of the concentration of the standard.
And (3) detection results:
as shown in FIG. 3, T4 was detected by the acridinium ester analog separation-free chemiluminescence immune competition method, and the minimum detection limit of T4 of PBS gradient dilution was 1ng/ml, which is basically the same as the detection sensitivity in serum. The linear range is 3 orders of magnitude.
Example 3
Horse radish peroxidase is coupled with clenbuterol small molecular marker BSA carrier protein (CLE-BSA), and Clenbuterol (CLE) monoclonal antibody is coupled with acridine ester analog for detection by an immune competition method. The following steps are carried out in sequence:
2. horse radish peroxidase coupled CLE-BSA
Mu.g of CLE-BSA was added to 1ml of 10mM carbonate buffer (pH8.0), 100. mu.g of horseradish peroxidase was added, and the mixture was stirred gently at 37 ℃ for 2 hours in the dark. Purifying the reaction solution by adding SUPERDEX200 gel filtration column, collecting the coupling product (HRP-CLE) of purified HRP and CLE-BSA protease, and storing at-20 deg.C with a storage solution (PBS + 0.05% Proclin-300).
CLE monoclonal antibody coupled acridinium ester analogs
1mg of the luminescent substrate acridinium ester analog (A) is dissolved in 2ml of DMF, 100. mu.l of the dissolved acridinium ester analog is added to 100. mu.l of CLE monoclonal antibody with the concentration of 10mg/ml, and then 800. mu.l of 0.05M boric acid buffer is added, and the reaction is performed for 1h at room temperature by rotation. Adding 1ml of the reaction solution into a dialysis bag with the molecular weight cutoff of 7000, dialyzing in a PBS buffer solution at 4 ℃ for 1 day, replacing the PBS buffer solution for 3 times during the dialysis, obtaining the CLE monoclonal antibody (CLE-A) connected with the acridine ester analog, and finally storing at-20 ℃ by using 1ml of a storage solution (PBS + 0.05% Proclin-300).
3. Detection of
Preparing an exciting liquid: 25mM tris (pH8.0), 1mM p-phenylphenol, 0.5mM EDTA, 0.2% Tween-20 and 5mM carbamide peroxide.
Preparing a background inhibitor: 25mM hydroxydiethylamine in deionized water.
CLE sample: CLE standards were diluted with PBS buffer (pH 7.4) to different concentrations of 100ng/ml, 10ng/ml, 1ng/ml, 0.1ng/ml, 0.01ng/ml, and stored at 4 ℃ as calibrators. And diluting the CLE standard substance to 100ng/ml, 10ng/ml, 1ng/ml, 0.1ng/ml and 0.01ng/ml by using CLE negative pig urine.
Sample adding detection: adopting a competition method, respectively diluting the HRP-CLE and CLE-A prepared by the method by 10000 times and 1500 times by using PBS buffer solution, respectively adding 100 mu l of HRP-CLE and CLE-A to a sample to be detected during detection, and additionally adding 10 mu l of background inhibitor into a transparent reaction tube. After incubation for 15min at 37 ℃ the assay was started immediately by adding 100. mu.l of excitation solution.
Establishing a corresponding CLE detection calculation formula according to the luminous signal value obtained by the calibration object of each concentration and the CLE concentration value corresponding to the luminous signal value, wherein the calculation formula comprises the following steps: y 2.94479-0.50971x (r)20.94310) where y represents the base 10 logarithm of the luminescence signal value and x represents the base 10 logarithm of the concentration of the standard.
And (3) detection results:
as shown in figure 4, the CLE is detected by the acridinium ester analogue separation-free chemiluminescence immune competition method, the minimum detection limit of the CLE diluted by PBS gradient is 0.01ng/ml, and the detection sensitivity is basically the same as that of the CLE in urine. The linear range is 3 orders of magnitude.
Example 4
Horse radish peroxidase is coupled with ractopamine small molecular marker BSA carrier protein (RAC-BSA), and Ractopamine (RAC) monoclonal antibody is coupled with acridine ester analogues for detection by an immune competition method. The following steps are carried out in sequence:
3. horse radish peroxidase-coupled RAC-BSA
Mu.g of RAC-BSA was added to 1ml of 10mM carbonate buffer (pH8.0), 100. mu.g of horseradish peroxidase was added thereto, and the mixture was stirred gently at 37 ℃ in the dark for 2 hours. Purifying the reaction solution by adding SUPERDEX200 gel filtration column, collecting the coupling product (HRP-RAC) of purified HRP and RAC-BSA substance enzyme, and storing at-20 deg.C with a storage solution (PBS + 0.05% Proclin-300).
RAC monoclonal antibody coupled acridinium ester analogs
1mg of the luminescent substrate acridinium ester analog (A) is dissolved in 2ml of DMF, 100. mu.l of the dissolved acridinium ester analog is added to 100. mu.l of RAC monoclonal antibody with the concentration of 10mg/ml, and then 800. mu.l of 0.05M boric acid buffer is added, and the reaction is performed for 1h at room temperature by rotation. Adding 1ml of the reaction solution into a dialysis bag with the molecular weight cutoff of 7000, dialyzing in PBS buffer at 4 ℃ for 1 day, replacing PBS buffer for 3 times during the dialysis, obtaining RAC monoclonal antibody (RAC-A) connected with acridine ester analogues, and finally storing at-20 ℃ by using 1ml of a storage solution (PBS + 0.05% Proclin-300).
3. Detection of
Preparing an exciting liquid: 25mM tris (pH8.0), 1mM p-phenylphenol, 0.5mM EDTA, 0.2% Tween-20 and 5mM carbamide peroxide.
Preparing a background inhibitor: 25mM hydroxydiethylamine in deionized water.
RAC samples: RAC standard substance is diluted with PBS buffer solution (PH 7.4) to different concentration values of 100ng/ml, 10ng/ml, 1ng/ml, 0.1ng/ml and 0.01ng/ml, and stored at 4 ℃ to be used as calibration substance. And diluting RAC standard substance with RAC negative pig urine to 100ng/ml, 10ng/ml, 1ng/ml, 0.1ng/ml and 0.01 ng/ml.
Sample adding detection: adopting a competition method, diluting the HRP-RAC and RAC-A prepared above by 18000 times and 1800 times respectively by PBS buffer solution, adding 100 mul of HRP-RAC and RAC-A to-be-detected samples respectively during detection, and adding 10 mul of background inhibitor into a transparent reaction tube. After incubation for 15min at 37 ℃ the assay was started immediately by adding 100. mu.l of excitation solution.
Establishing a corresponding RAC detection calculation formula according to the luminous signal value obtained by the calibration object of each concentration and the corresponding RAC concentration value, wherein the RAC detection calculation formula comprises the following steps: y 3.07006-0.45831x (r)20.92930) where y represents the base 10 logarithm of the luminescence signal value and x represents the base 10 logarithm of the concentration of the standard.
And (3) detection results:
as shown in FIG. 5, the RAC is detected by the acridinium ester analogue separation-free chemiluminescence immune competition method, and the lowest detection limit of the RAC diluted by PBS gradient is 0.01ng/ml, which is basically the same as the detection sensitivity in urine. The linear range is 3 orders of magnitude.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (10)

1. The application of the acridinium ester analogue in preparing a acridinium ester analogue labeled antibody; the molecular structural formula of the acridine ester analog is shown as a formula I;
Figure FDA0003158053480000011
2. the application of the acridinium ester analog in preparing a competitive detection kit; the molecular structural formula of the acridine ester analog is shown as a formula I;
Figure FDA0003158053480000012
3. a detection kit by a competition method comprises an acridinium ester analogue labeled antibody, an enzyme-labeled antigen and an excitation liquid;
the molecular structural formula of the acridine ester analog is shown as a formula I;
Figure FDA0003158053480000013
4. the competitive assay kit of claim 3 further comprising a background inhibitor.
5. The competitive assay kit of claim 4, wherein the background inhibitor comprises an aqueous solution of hydroxydiethylamine at a molar concentration of 20 to 30 mM.
6. The competitive assay kit of claim 3 wherein the trigger solution comprises the following components in concentrations: 20 to 30mM tris, 0.5 to 1.5mM p-phenylphenol, 0.3 to 0.8mM EDTA, 0.1 to 0.3% Tween-20 and 3 to 8mM carbamide peroxide; the pH value of the tris is 7.8-8.2.
7. The competitive assay kit of claim 3 wherein the enzyme-labeled antigen comprises a horseradish peroxidase-labeled antigen.
8. The competition method detection kit according to claim 3, wherein the competition method detection kit comprises a T3 detection kit, a T4 detection kit, a clenbuterol detection kit or a ractopamine detection kit.
9. The competition method detection kit according to claim 3, wherein the acridinium ester analog labeled antibody comprises an acridinium ester analog labeled T3 monoclonal antibody, an acridinium ester analog labeled T4 monoclonal antibody, an acridinium ester analog labeled clenbuterol monoclonal antibody or an acridinium ester analog labeled ractopamine monoclonal antibody.
10. A competitive assay method for non-diagnostic purposes comprising the steps of:
1) labeling the antibody with an acridinium ester analog;
2) mixing the antibody marked with the acridine ester analog, the enzyme-labeled antigen and a sample to be detected, and incubating at 35-40 ℃ for 12-15 min to obtain a reaction solution;
3) adding an excitation liquid into the reaction liquid, detecting a luminescent signal generated within 1 second after the excitation liquid is added, and obtaining the antigen concentration in the sample to be detected according to a standard curve and the luminescent signal;
the standard curve is obtained by establishing a luminous signal value obtained by a calibration object with gradient concentration and a concentration value corresponding to the calibration object;
the molecular structural formula of the acridine ester analog is shown as a formula I;
Figure FDA0003158053480000021
CN202110783301.6A 2021-07-12 2021-07-12 Application of acridinium ester analogue in preparation of acridinium ester analogue labeled antibody Withdrawn CN113533735A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110783301.6A CN113533735A (en) 2021-07-12 2021-07-12 Application of acridinium ester analogue in preparation of acridinium ester analogue labeled antibody

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110783301.6A CN113533735A (en) 2021-07-12 2021-07-12 Application of acridinium ester analogue in preparation of acridinium ester analogue labeled antibody

Publications (1)

Publication Number Publication Date
CN113533735A true CN113533735A (en) 2021-10-22

Family

ID=78098510

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110783301.6A Withdrawn CN113533735A (en) 2021-07-12 2021-07-12 Application of acridinium ester analogue in preparation of acridinium ester analogue labeled antibody

Country Status (1)

Country Link
CN (1) CN113533735A (en)

Similar Documents

Publication Publication Date Title
US20200309770A1 (en) Chemiluminescence immunoassay kit for adiponectin, and preparation method and use thereof
CN110862881B (en) Special cleaning solution or diluent for full-automatic chemiluminescence determinator and preparation method thereof
CN109917134B (en) Calibrator stabilizer, detection kit for determining C peptide and detection method
CN113419059B (en) Blocking agent for chemiluminescent immunoassay method
CN111323577A (en) Anti-cardiolipin antibody IgG magnetic particle chemiluminescence immunoassay kit
CN110988368A (en) Free thyroxine luminescence immunoassay kit and preparation method thereof
CN112285353A (en) Method for improving anti-biotin interference capability and sensitivity of chemiluminescence kit of streptavidin-biotin reaction system
CN111175494A (en) Thyroglobulin antibody detection kit and use method thereof
CN113933506A (en) Magnetic particle chemiluminescence detection kit for determining content of human chitinase 3-like protein 1(CHI3L1)
CN109655626A (en) A kind of detection kit and its method of complete homogeneous determination glucagon
CN111007266A (en) Chemiluminescence quantitative detection kit for detecting B-type natriuretic peptide in blood plasma
CN113238055A (en) Kit for detecting procalcitonin by using space proximity chemiluminescence method, and detection method and application thereof
CN113640511A (en) Magnetic particle electrochemiluminescence kit
CN113049805A (en) Antibody compound, preparation method thereof and detection kit
CN113533735A (en) Application of acridinium ester analogue in preparation of acridinium ester analogue labeled antibody
CN116519945A (en) Magnetic particle chemiluminescence detection kit for rapidly determining content of soluble ST2
CN111024940B (en) Time-resolved fluorescence immunoassay method based on gold magnetic particles
CN114778816A (en) Acridinium ester labeling compound and preparation method thereof
CN110878296B (en) High-sensitivity HRP enzyme and preparation method and application thereof
CN113219175A (en) Human growth differentiation factor 15 chemiluminescence immunoassay kit and preparation method thereof
JP3228791B2 (en) Measuring method of antigen or antibody in sample
CN112903664A (en) Enzymatic chemiluminescence substrate liquid of alkaline phosphatase
CN111381046A (en) Calprotectin chemiluminescence immunoassay kit and preparation method thereof
CN117054671B (en) Dissociation method of folic acid detection sample and folic acid detection method
CN101377499A (en) Nerve specificity olefinic alcohol enzyme chemiluminescence immune analysis determination reagent kit and preparing method thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
WW01 Invention patent application withdrawn after publication

Application publication date: 20211022

WW01 Invention patent application withdrawn after publication