CN114280289B - Magnetic particle chemiluminescence detection kit and detection method thereof - Google Patents
Magnetic particle chemiluminescence detection kit and detection method thereof Download PDFInfo
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Abstract
The invention relates to a magnetic particle chemiluminescence detection kit and a detection method thereof, wherein the kit comprises an acridinium ester labeled antibody, a capture antibody, magnetic beads and an acridinium ester antibody. According to the invention, through adopting the acridine ester antibody, the nonspecific adsorption of the acridine ester on the magnetic beads or the inner wall of the container is reduced, the nonspecific adsorption signal value of the acridine ester is effectively reduced by more than 60%, and the sensitivity of the chemiluminescent reaction is improved and the practicability is realized.
Description
Technical Field
The invention belongs to the field of medical detection, and mainly relates to a magnetic particle chemiluminescence detection kit and a detection method thereof.
Background
Chemiluminescent immunoassay (chemiluminescence immunoassay, CIA) can be classified into three types, direct chemiluminescent immunoassay and chemiluminescent enzyme immunoassay, and electrochemiluminescence, depending on the luminescent substance. Wherein the direct chemiluminescent immunoassay process does not need the catalysis of enzyme and directly participates in the luminescent reaction.
The chemiluminescent agent used in the direct luminescence immunoassay process has a luminescent group on its chemical structure, so that the process does not need the catalysis of enzyme to directly carry out luminescence reaction. The chemiluminescent agent directly marks the detection antibody, reacts with the sample to be detected and the capture antibody coated by the magnetic beads, separates a complex formed by the capture antibody coated by the magnetic beads, the sample to be detected and the marked detection antibody through a magnetic field, and then adds a luminescence promoter into the complex to carry out luminescence reaction, and carries out quantitative or qualitative detection on the detection of the luminescence intensity. The most commonly used chemiluminescent agent is the acridinium ester.
Acridinium esters are a general name of acridinium salts, refer to chemical substances used as chemiluminescent markers, are different in series, are low in background luminescence, can be used for detecting low-concentration or trace-concentration samples in a reaction, are luminescent agents with high luminous efficiency, have the characteristics of rapid luminescence, high luminous value, sensitive detection and the like, are widely accepted by the immunodetection field, and are increasingly applied to immunodetection reagents.
However, acridinium esters have a difficulty in application in chemiluminescent immunoassay, in that there is a certain nonspecific adsorption of acridinium esters, and the nonspecific adsorption mainly comes from free acridinium esters which are not combined with detection antibodies in acridinium ester labeled detection antibody solution, and the free acridinium esters are adsorbed on magnetic beads or reaction inner walls, so that the background is increased, and the detection accuracy is reduced.
There is no method for reducing the nonspecific adsorption of acridine ester, and there is an urgent need in the art to develop a method for reducing the nonspecific adsorption of acridine ester.
Disclosure of Invention
When the sample to be tested is incubated with the magnetic bead working solution coated with the capture antibody and the detection antibody working solution marked with the acridine ester, a complex of 'the magnetic bead coated with the capture antibody-the sample to be tested-the detection antibody marked with the acridine ester' is formed, but because free acridine ester which is not combined with the detection antibody exists in the working solution marked with the acridine ester, after the acridine ester antibody is added into the complex, the acridine ester antibody can be competitively combined with the magnetic bead or the inner wall of the container, the incidence rate of nonspecific combination of the free acridine ester and the magnetic bead or the inner wall of the container is reduced, the background is reduced, the sensitivity in the detection process is improved, and the linear performance of the reagent is not affected by the addition of the acridine ester antibody.
The invention provides a magnetic particle chemiluminescence detection kit which comprises an acridinium ester labeled antibody, a capture antibody and magnetic beads.
In some embodiments, the acridinium ester-labeled antibody is an acridinium ester-labeled PCT detection antibody and the capture antibody is a PCT capture antibody.
The invention also provides a magnetic particle chemiluminescence detection method, which comprises the steps of adding a sample to be detected into a reaction cup in the sample detection process, and adding a capture antibody, a magnetic bead, an acridine ester antibody and an acridine ester labeled antibody into the reaction cup.
In some embodiments, the capture antibody is a coated biotin-labeled PCT capture antibody, the magnetic beads are streptavidin magnetic bead working fluid, and the acridinium ester-labeled antibody is an acridinium ester-labeled PCT detection antibody.
In some embodiments, the structure of the acridinium ester in the acridinium ester-labeled antibody is as follows:
in some embodiments, the acridinium ester antibody is selected from a monoclonal antibody or a polyclonal antibody, preferably a monoclonal antibody.
In some embodiments, the acridinium ester antibody is a monoclonal antibody, the VL CDR1, CDR2 and CDR3 of which have the amino acid sequences of SEQ ID NO:1-3, respectively, and the VH CDR1, CDR2 and CDR3 of which have the amino acid sequences of SEQ ID NO:4-6, respectively.
In some embodiments, the molar ratio of the acridinium ester antibody to the acridinium ester used for labeling is selected from 0.1 to 5:1, preferably 0.5 to 4:1, more preferably 1 to 3:1, more preferably 1:1, 1.5:1, 2:1, 2.5:1, 3:1, most preferably 2:1. The invention also provides an acridinium ester antibody which is characterized in that VL CDR1, CDR2 and CDR3 respectively have the amino acid sequences of SEQ ID NO. 1-3, and VH CDR1, CDR2 and CDR3 respectively have the amino acid sequences of SEQ ID NO. 4-6.
The invention also provides the application of the acridinium ester antibody in preparing a magnetic particle chemiluminescence detection kit or in a magnetic particle chemiluminescence detection method.
In some embodiments, a buffer is also included in the kit. In some embodiments, the buffer is selected from the group consisting of Phosphate Buffered Saline (PBS), 2-morpholinoethanesulfonic acid buffer (MES), tris-HCl, hydroxyethylpiperazine ethanesulfuric acid solution buffer (HEPES). In some embodiments, the concentration of the buffer is selected from 0.01mol/L to 0.1mol/L, preferably from 0.01mol/L to 0.05mol/L, more preferably from 0.01mol/L, 0.02mol/L, 0.03mol/L, 0.04mol/L, 0.05mol/L. In some embodiments, the pH of the buffer is selected from 6.0 to 7.5, more preferably from 7.0, 7.1, 7.2, 7.3, 7.4, 7.5. In some embodiments, the pH of the buffer is 7.4.
In some embodiments, the acridinium ester antibody is a monoclonal antibody, the VL CDR1, CDR2 and CDR3 of which have the amino acid sequences of SEQ ID NO 1-3, respectively, and the VH CDR1, CDR2 and CDR3 of which have the amino acid sequences of SEQ ID NO 4-6, respectively; the buffer solution is PBS buffer solution, the concentration of the buffer solution is 0.01mol/L, and the pH value is 7.4.
Definition:
the term "capture antibody" as used herein refers to an antibody that is capable of specifically recognizing and binding to an antigen to be tested from the test object. The term "detection antibody" refers to another antibody capable of specifically recognizing an antigen to be detected, which specifically recognizes different epitopes on the same antigen molecule to be detected with a capture antibody, and the detection antibody labels acridinium ester, thereby realizing quantitative or qualitative detection of the antigen to be detected.
The term "acridinium ester for labeling" as used herein refers to an acridinium ester for labeling a detection antibody, which includes free acridinium esters, acridinium esters bound to detection antibodies. "free acridinium ester" refers to an acridinium ester that is not bound to a detection antibody.
The term "inner wall of the container" as used in the present invention refers to the inner wall of a reaction cup used in conjunction with a chemiluminescent immunoassay analyzer. The reaction cup refers to a reaction container matched with the chemiluminescent immunoassay analyzer.
The term "protein A affinity column" as used herein refers to an affinity column which has become a widely used affinity column for purifying antibodies, and which can separate and purify antibodies of various mammalian subtypes or genetically engineered recombinant proteins comprising Fc fragments of antibodies from ascites, serum and cell culture supernatants or cell extracts.
The beneficial effects are that:
1: effectively reducing the non-specific adsorption signal value of the acridinium ester by more than 60 percent;
2: the sensitivity of the chemiluminescent reaction is improved and the practicability is realized.
Detailed Description
Materials:
NaCl、Na 2 HPO 4 ·12H 2 O、NaHPO 4 ·2H 2 o, hydrochloric acid (all from China national medicine group)
Bovine serum albumin (from Merck, cat# A1933-25G)
Carrier protein Keyhole Limpet Hemocyanin (KLH) (from Merck, cat# F5881-6X)
Freund's complete adjuvant (from Sigma, cat# F5881-6X)
Substrate liquid (ThermoFisher Scientific, cargo number 34021)
Myeloma cell (from Punuocele, cat# CL-0217)
Acridine ester (from Herison, cat HS-12000002)
Streptavidin magnetic bead kit (from ThermoFisher Scientific, cat No. 88017)
Biotin coupling kit (from ThermoFisher Scientific, cat 21425)
Acridine ester chemiluminescent labeling kit (from Helison, cat No. HS-12000002)
PCT Capture antibody (from Nuo Wei Zan, cat# RM 3321)
PCT detection antibody (from Nuo Wei Zan, cat# RM 3322)
Acid-base excitation liquid: the following chemiluminescent immunoassay analyzer was purchased and obtained as a gift
Sample Procalcitonin (PCT) (from a plasma sample containing PCT)
Instrument:
multifunctional enzyme label instrument (from TECAN)
Chemiluminescent immunoassay analyzer (from Kesi mai instrument model SMART 500)
Example 1: acquisition of acridinium ester antibodies
Example 1.1: preparation of acridinium ester artificial antigen
(1) PBS solution of 0.01mol/L was prepared: a clean beaker was taken, 500mL of pure water was added thereto, and 8.78g of NaCl and 5.8g of Na were added thereto 2 HPO 4 ·12H 2 O and 0.59g of NaHPO 4 ·2H 2 O, adjust pH to 7.4 with appropriate amount of hydrochloric acid, then fix volume to 1000mL.
(2) 200 mu L of the PBS solution in the step (1) is taken to enter a test tube, 200 mu g of the carrier protein Keyhole Limpet Hemocyanin (KLH) and 100 mu M of acridine ester are sequentially added into the test tube, the mixture is uniformly mixed, the reaction is carried out for 1h at room temperature, the mixture is purified for 5 times through a 50KD ultrafiltration tube, the purified product is collected into a 1.5mL EP tube, the acridine ester-carrier protein keyhole limpet hemocyanin coupling is completed, and the mixture is stored at 4 ℃ for standby.
Example 1.2: immunization of mice with prepared acridinium ester-carrier protein keyhole limpet hemocyanin conjugates
(1) The prepared acridinium ester-carrier protein keyhole limpet hemocyanin conjugate is mixed with Freund's complete adjuvant in equal quantity, and vortex is carried out for 30min to completely emulsify the acridinium ester-carrier protein keyhole limpet hemocyanin conjugate. The primary immunization was completed by injecting the emulsion into healthy BALB/c mice at a dose of 80. Mu.g/dose by subcutaneous multipoint injection.
(2) After one month, the acridinium ester-carrier protein keyhole limpet hemocyanin conjugate was mixed with Freund's incomplete adjuvant in equal amounts, vortexed for 30min to completely emulsify, and the emulsion was injected into healthy BALB/c mice at a dose of 50 μg/dose by subcutaneous multipoint injection. Secondary immunization is completed.
(3) And carrying out third boosting immunization on the third week after the second immunization, wherein the step of boosting immunization is the same as the second immunization.
Example 1.3 cell fusion and screening
The spleen of the mouse is taken aseptically, spleen cells are extracted, and then the spleen cells are fused with myeloma cells by an electrofusion method. The hybridoma cells were selected for culture using HAT/HT selection medium. And taking cell supernatant after culturing, screening positive cell strains by adopting an indirect ELISA method, and carrying out multiple subcloning screening culture to obtain hybridoma cell strains capable of stably secreting the acridinium ester antibody.
Example 1.4 purification of antibody preparation
The hybridoma cell strain obtained in the example 1.3 is injected into a mouse body by adopting an in-vivo induction method to prepare ascites, and the titer is measured by an indirect ELISA method after the ascites is collected. Antibody purification was performed using a protein a antibody purification column to obtain an acridinium ester antibody, and the sequence of the antibody was determined by a polypeptide sequencing method, and the light chain and heavy chain CDR regions of the acridinium ester antibody were identified as shown in the following table:
TABLE 1 light and heavy chain CDRs of acridinium ester antibodies
| CDR | The corresponding SEQ ID NO. | Sequence(s) |
| VL CDR1 | 1 | ENIYTN |
| VL CDR2 | 2 | GAT |
| VL CDR3 | 3 | QHFWGTPFT |
| VH CDR1 | 4 | GYSITSDYA |
| VH CDR2 | 5 | ISYSGRT |
| VH CDR3 | 6 | ASAWDY |
Example 2: working solution for preparing streptavidin magnetic beads
And (3) operating according to the instruction of the streptavidin magnetic bead kit, finally obtaining 1mg/mL streptavidin magnetic bead working solution, and storing the working solution in a refrigerator at 4 ℃ for later use.
Example 3: working solution for preparing biotin-labeled PCT (PCT) capture antibody
According to the biotin coupling kit instructions operation, finally obtain 0.3ug/mL labeled biotin PCT capture antibody, and store in 4 ℃ refrigerator for use.
Example 4: working solution for preparing acridinium ester marked PCT detection antibody
(1) PCT detection antibodies were acridine ester labeled according to the acridine ester chemiluminescent labeling kit instructions, wherein the acridine ester concentration used to label the detection antibodies was 0.5mmol/L, using 10. Mu.L of acridine ester.
(2) After the labeling is completed, the concentration of the PCT detection antibody of the labeled acridinium ester is determined by an enzyme-labeling instrument, then the detection antibody of the labeled acridinium ester is diluted to 0.3ug/mL by a buffer solution in an acridinium ester chemiluminescence labeling kit, and the detection antibody is stored in a refrigerator at 4 ℃ for standby.
Example 5: detecting a sample
(1) Experiments were performed with molar ratios of acridinium ester antibody to acridinium ester used for labeling of 0.5:1, 1:1, 2:1, 4:1, respectively.
(2) Adding 50 mu L of a sample to be detected into a reaction cup, adding 50 mu L of a coated biotin-labeled PCT capture antibody, 50 mu L of a streptavidin magnetic bead working solution, 50 mu L of an acridine ester antibody and 50 mu L of an acridine ester-labeled PCT detection antibody working solution into the reaction cup in the following sequence, incubating for 10min at 37 ℃, and cleaning.
(3) The reaction cup was placed on a chemiluminescent immunoassay analyzer for testing at a wavelength of 430nm.
The results of the detection are shown in tables 2, 3, 4 and 5 (the molar ratio of the acridinium ester antibody to the acridinium ester used for labeling is 0.5:1, 1:1, 2:1 and 4:1, respectively).
Table 2 (0.5:1):
| signal value of control group | Experimental group signal values | Relative amplitude of variation | |
| S0 | 5000 | 4901 | -2.0％ |
| S1 | 21871 | 21035 | -3.8％ |
| S2 | 58941 | 55981 | -5.0％ |
| S3 | 286794 | 281561 | -1.8％ |
| S4 | 1286941 | 1305481 | 1.4％ |
Table 3 (1:1):
| signal value of control group | Experimental group signal values | Relative amplitude of variation | |
| S0 | 5000 | 3038 | -39.2％ |
| S1 | 21871 | 20632 | -5.7％ |
| S2 | 58941 | 55678 | -5.5％ |
| S3 | 286794 | 279675 | -2.5％ |
| S4 | 1286941 | 1263872 | -1.8％ |
Table 4 (2:1):
| signal value of control group | Experimental group signal values | Relative amplitude of variation | |
| S0 | 5000 | 1500 | -70.0％ |
| S1 | 21871 | 20056 | -8.3％ |
| S2 | 58941 | 56816 | -3.6％ |
| S3 | 286794 | 279613 | -2.5％ |
| S4 | 1286941 | 1324872 | 2.9％ |
Table 5 (4:1):
| signal value of control group | Experimental group signal values | Relative amplitude of variation | |
| S0 | 5000 | 1602 | -68.0％ |
| S1 | 21871 | 18620 | -14.9％ |
| S2 | 58941 | 48950 | -17.0％ |
| S3 | 286794 | 214586 | -25.2％ |
| S4 | 1286941 | 996542 | -22.6％ |
The control group indicates that no acridine ester antibody was added, and the experimental group indicates that the corresponding molar amount of acridine ester antibody was added. S0 indicates that no PCT antigen was added, S1 indicates that 0.5ng/mL of PCT antigen was added, S2 indicates that 2ng/mL of PCT antigen was added, S3 indicates that 10ng/mL of PCT antigen was added, and S4 indicates that 50ng/mL of PCT antigen was added.
Analysis of results
Under the condition of S0, the generated signal is nonspecific adsorption, and the reduction amplitude of the nonspecific adsorption of the experimental group compared with the control group is represented by 'relative amplitude' in a table. The molar ratio of acridinium ester antibody to acridinium ester used for labeling was 0.5:1, 1:1, 2:1, 4:1, respectively, and the nonspecific adsorption was reduced by 2.0%, 39.2%, 70.0%, 68.0%, respectively.
Meanwhile, the larger the value of S1 (or S2 or S3 or S4)/S0, the better the sensitivity. When the molar ratio of acridinium ester antibody to acridinium ester is 0.5:1, the S1/S0=4.37 of the control group and the S1/S0=4.29 of the experimental group; when the molar ratio of acridinium ester antibody to acridinium ester used for labeling is 1:1, S1/s0=4.37 in the control group, S1/s0=6.79 in the experimental group; when the molar ratio of acridinium ester antibody to acridinium ester used for labeling is 2:1, S1/s0=4.37 in the control group, S1/s0=13.37 in the experimental group; when the molar ratio of acridinium ester antibody to acridinium ester is 4:1, the S1/S0=4.37 of the control group and the S1/S0=11.62 of the experimental group. As can be seen from the results, the experimental group and the control group were the most sensitive when the molar ratio was 2:1, indicating that the sensitivity was significantly improved. When acridinium ester antibodies: when the molar ratio of the acridinium ester used for labeling is 0.5:1, 1:1 and 2:1, the corresponding relative amplitude of S1-S4 is within 10%, which indicates that the acridinium ester antibody does not influence the linearity and other performances of the reagent in the experiment of detecting the antigen by a chemiluminescence method, and has good practicability, but when the acridinium ester antibody: the molar ratio of the acridinium esters used for labeling is 4:1, and the "relative amplitude" corresponding to S1-S4 is all outside 10%, which indicates that at this ratio, the acridinium ester antibodies affect the linear function of the reagent in the chemiluminescent assay for antigen detection.
Sequence listing
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<120> a magnetic particle chemiluminescence detection kit and a detection method thereof
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Claims (14)
1. The magnetic particle chemiluminescence detection kit comprises an acridinium ester labeled antibody, a capture antibody and magnetic beads, and is characterized by further comprising an acridinium ester antibody, wherein the acridinium ester antibody is combined with free acridinium ester, the free acridinium ester is the acridinium ester which is not combined with a detection antibody, and the acridinium ester labeled antibody is the acridinium ester labeled detection antibody.
2. The kit of claim 1, wherein the acridinium ester-labeled antibody is an acridinium ester-labeled PCT detection antibody and the capture antibody is a PCT capture antibody.
3. A magnetic particle chemiluminescence detection method is characterized by comprising the steps of adding a sample to be detected into a reaction cup in a sample detection process, and adding a capture antibody, a magnetic bead, an acridine ester antibody and an acridine ester labeled antibody into the reaction cup, wherein the acridine ester antibody is combined with free acridine ester, the free acridine ester is an acridine ester which is not combined with a detection antibody, and the acridine ester labeled antibody is an acridine ester labeled detection antibody.
4. The method of claim 3, wherein the capture antibody is a coated biotin-labeled PCT capture antibody, the magnetic beads are streptavidin magnetic bead working fluid, and the acridinium ester-labeled antibody is an acridinium ester-labeled PCT detection antibody.
5. The kit according to claim 1 or the detection method according to claim 3, wherein the structure of the acridinium ester in the acridinium ester-labeled antibody is as follows:
6. the kit according to claim 1 or the detection method according to claim 3, wherein the acridinium ester antibody is selected from a monoclonal antibody or a polyclonal antibody.
7. The kit of claim 1 or the detection method of claim 3, wherein the acridinium ester antibody is a monoclonal antibody.
8. The kit according to claim 1 or the detection method according to claim 3, wherein the acridinium ester antibody is a monoclonal antibody, its VL CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs 1 to 3, respectively, and its VH CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NOs 4 to 6, respectively.
9. The kit according to claim 1 or the detection method according to claim 3, wherein the molar ratio of the acridinium ester antibody to the acridinium ester used for labeling is selected from 0.1 to 5:1.
10. The kit according to claim 1 or the detection method according to claim 3, wherein the molar ratio of the acridinium ester antibody to the acridinium ester used for labeling is selected from 0.5 to 4:1.
11. The kit according to claim 1 or the detection method according to claim 3, wherein the molar ratio of the acridinium ester antibody to the acridinium ester used for labeling is selected from 1 to 3:1.
12. The kit according to claim 1 or the detection method according to claim 3, wherein the molar ratio of the acridinium ester antibody to the acridinium ester used for labeling is 2:1.
13. An acridinium ester antibody, characterized in that VL CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NO 1-3, respectively, and VH CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NO 4-6, respectively.
14. Use of the acridinium ester antibody of claim 13 in the preparation of a magnetic particle chemiluminescent detection kit or in a magnetic particle chemiluminescent detection method.
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