CN114280289A - 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, a magnetic bead and an acridinium ester antibody. By adopting the acridinium ester antibody, the invention reduces the nonspecific adsorption of the acridinium ester on the magnetic beads or the inner wall of the container, effectively reduces the value of the acridinium ester nonspecific adsorption signal by more than 60 percent, improves the sensitivity of the chemiluminescence reaction and has practicability.
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
Chemiluminescence Immunoassay (CIA) can be classified into direct chemiluminescence immunoassay and chemiluminescence enzyme immunoassay, and electrochemiluminescence, according to the difference of luminescent substances. Wherein the direct chemiluminescence immunoassay process does not need the catalysis of enzyme and directly participates in the luminescence reaction.
The chemical structure of the chemiluminescent reagent used in the direct luminescence immunoassay process has a luminescent group, so that the process does not need the catalytic action of enzyme and directly carries out luminescence reaction. The chemiluminescence reagent directly marks a detection antibody, the detection antibody reacts with a sample to be detected and a capture antibody coated by magnetic beads, a complex formed by the capture antibody coated by the magnetic beads, the sample to be detected and the marked detection antibody is separated through a magnetic field, then a luminescence promoter is added into the complex for luminescence reaction, and quantitative or qualitative detection is carried out through detection of luminescence intensity. The most commonly used chemiluminescent agent is an acridinium ester.
Acridinium ester is a general term of acridinium salt, refers to a chemical substance used as a chemiluminescent marker, has different series, generally has low background luminescence, can be used for detecting samples with low concentration or trace concentration in reaction, is a luminescent agent with high luminescent efficiency, has the characteristics of rapid luminescence, high luminescent value, sensitive detection and the like, is widely accepted in the field of immunoassay, and is increasingly applied to immunoassay reagents.
However, the acridinium ester has a difficulty in application in chemiluminescence immunoassay, and the difficulty is that the acridinium ester has a certain non-specific adsorption, the non-specific adsorption mainly comes from free acridinium ester which is not combined with a detection antibody in an acridinium ester labeled detection antibody solution, and the free acridinium ester can be adsorbed on magnetic beads or reaction inner walls, so that the background is increased, and the detection accuracy is reduced.
At present, no method capable of reducing the nonspecific adsorption of the acridinium ester exists, and the development of a method for reducing the nonspecific adsorption of the acridinium ester is urgently needed in the field.
Disclosure of Invention
When a sample to be detected is incubated with magnetic bead working solution coated with a capture antibody and detection antibody working solution marked with acridinium ester, a compound of 'magnetic bead coated with the capture antibody-sample to be detected-detection antibody marked with acridinium ester' is formed, but because free acridinium ester which is not combined with the detection antibody exists in the working solution marked with the detection antibody of acridinium ester, after the acridinium ester antibody is added into the compound, the acridinium ester antibody can be combined with the free acridinium ester competitively with the inner wall of the magnetic bead or the container, the occurrence rate of non-specific combination of the free acridinium ester and the inner wall of the magnetic bead or the container is reduced, the background is reduced, the sensitivity in the detection process is improved, and the linear performance of a reagent is not influenced by the addition of the acridinium ester antibody.
The invention provides a magnetic particle chemiluminescence detection kit which comprises an acridinium ester labeled antibody, a capture antibody and magnetic beads, and is characterized by further comprising the acridinium ester antibody.
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 the reaction cup in the sample detection process, and adding the capture antibody, the magnetic bead, the acridinium ester antibody and the acridinium ester labeled antibody into the reaction cup.
In some embodiments, the capture antibody is a biotin-labeled PCT capture antibody coated bead, the bead is a streptavidin bead working solution, 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 having VL CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs 1-3, respectively, and VH CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs 4-6, respectively.
In some embodiments, the molar ratio of the acridinium ester antibody to the acridinium ester used for labeling is selected from 0.1-5: 1, preferably 0.5-4: 1, more preferably 1-3: 1, more preferably 1:1, 1.5:1, 2:1, 2.5:1, 3:1, and most preferably 2: 1. The invention also provides an acridinium ester antibody, which is characterized in that VL CDR1, CDR2 and CDR3 respectively have amino acid sequences of SEQ ID NO. 1-3, and VH CDR1, CDR2 and CDR3 respectively have amino acid sequences of SEQ ID NO. 4-6.
The invention also provides application of the acridinium ester antibody in preparation of a magnetic particle chemiluminescence detection kit or 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 Phosphate Buffered Saline (PBS), 2-morpholinoethanesulfonic acid buffer (MES), Tris-HCl buffer, hydroxyethylpiperazine ethanesulfonic acid solution buffer (HEPES). In some embodiments, the buffer has a concentration selected from the group consisting of 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, 0.05 mol/L. In some embodiments, the pH of the buffer is selected from 6.0-7.5, 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 having VL CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs 1-3, respectively, and having VH CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs 4-6, respectively; the buffer solution is PBS buffer solution, the concentration of the PBS buffer solution is 0.01mol/L, and the pH value is 7.4.
Defining:
the term "capture antibody" as used herein refers to an antibody that specifically recognizes and binds to an antigen to be detected from an analyte. The term "detection antibody" refers to another antibody capable of specifically recognizing the antigen to be detected, which and the capture antibody respectively specifically recognize different epitopes on the same antigen molecule to be detected, and the detection antibody is labeled with acridinium ester, thereby realizing the quantitative or qualitative detection of the antigen to be detected.
The term "acridinium ester for labeling" as used herein refers to acridinium ester for labeling a detection antibody, which includes free acridinium ester, acridinium ester bound to a detection antibody. "free acridinium ester" refers to acridinium ester that is not bound to a detection antibody.
The term "inner wall of the container" as used herein refers to the inner wall of a reaction cup used in conjunction with a chemiluminescent immunoassay analyzer. The term "reaction cup" refers to a reaction vessel used in conjunction with a chemiluminescent immunoassay analyzer.
The term "ProteinA affinity column" as used herein refers to an affinity column which has become widely used for the purification of antibodies, and can separate and purify various subtypes of antibodies of mammals or genetically engineered recombinant proteins containing Fc fragments of antibodies from ascites, serum and cell culture supernatants or cell extracts.
Has the advantages that:
1: effectively reducing the value of the acridinium ester non-specific adsorption signal by over 60 percent;
2: the sensitivity of the chemiluminescence reaction is improved and the method has practicability.
Detailed Description
Materials:
NaCl、Na2HPO4·12H2O、NaHPO4·2H2o, hydrochloric acid (all from the Chinese medicine group)
Bovine serum albumin (from Merck, cat # A1933-25G)
Keyhole Limpet Hemocyanin (KLH) (from Merck, cat # F5881-6X)
Freund's complete adjuvant (from Sigma, cat # F5881-6X)
Substrate solution (ThermoFisher Scientific, cat 34021)
Myeloma cell (from Punuisai, cat # CL-0217)
Acridine ester (from Herrison, cat # HS-12000002)
Streptavidin magnetic bead kit (from ThermoFisher Scientific, cat # 88817)
Biotin coupling kit (from ThermoFisher Scientific, cat. No. 21425)
Acridinium ester chemiluminescent labeling kit (from Herrison, cat # HS-12000002)
PCT capture antibody (from Nodezan, cat # RM3321)
PCT detection antibody (from Nodezan, cat # RM3322)
Acid-base excitation liquid: purchase the following chemiluminescence immunoassay analyzer
Sample Procalcitonin (PCT) (from plasma samples containing PCT)
The instrument comprises the following steps:
multifunctional enzyme mark instrument (from TECAN)
Chemiluminescence immunoassay analyzer (from Kesimei, instrument model SMART500)
Example 1: obtaining acridinium ester antibodies
Example 1.1: preparation of acridinium ester artificial antigen
(1) Preparing 0.01mol/L PBS solution: a clean beaker was taken, 500mL of pure water was added thereto, and 8.78g of NaCl and 5.8g of Na were further added thereto2HPO4·12H2O and 0.59g of NaHPO4·2H2And O, adjusting the pH to 7.4 by using a proper amount of hydrochloric acid, and then metering to 1000 mL.
(2) And (2) putting 200 mu L of the PBS solution in the step (1) into a test tube, sequentially adding 200 mu g of carrier protein Keyhole Limpet Hemocyanin (KLH) and 100 mu M of acridinium ester into the test tube, uniformly mixing, reacting at room temperature for 1h, purifying for 5 times through a 50KD ultrafiltration tube, collecting the purified product into a 1.5mL EP tube, completing acridinium ester-carrier protein keyhole limpet hemocyanin coupling, and storing at 4 ℃ for later use.
Example 1.2: immunization of mice with prepared acridinium ester-carrier protein keyhole limpet hemocyanin conjugate
(1) The prepared acridinium ester-carrier protein keyhole limpet hemocyanin conjugate is mixed with Freund's complete adjuvant in equal amount, and vortexed for 30min to emulsify completely. The primary immunization was performed by injecting the emulsion into healthy BALB/c mice at a dose of 80. mu.g/mouse by subcutaneous multi-point injection.
(2) One month later, 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. mu.g/mouse by subcutaneous multi-point injection. Thus completing the secondary immunization.
(3) And performing a third boosting immunization in the third week after the second immunization, wherein the boosting immunization step is the same as the second immunization.
Example 1.3 cell fusion and screening
Spleen cells of mice are aseptically taken, and are extracted and then fused with myeloma cells by an electric fusion method. And (4) screening hybridoma cells by using HAT/HT selective medium for culture. And (3) taking cell supernatant after culture, screening positive cell strains by adopting an indirect ELISA method, and obtaining the hybridoma cell strain capable of stably secreting the acridinium ester antibody through multiple times of subcloning, screening and culturing.
Example 1.4 purification of antibody preparation
The hybridoma cell line obtained in example 1.3 was injected into a mouse by an in vivo induction method to prepare ascites, and the titer was measured by an indirect ELISA method after the ascites was collected. Purifying the antibody by a ProteinA antibody purification column to obtain an acridinium ester antibody, determining the sequence of a monoclonal antibody by a polypeptide sequence determination method, and identifying that the sequences of CDR regions of a light chain and a heavy chain of the acridinium ester antibody are shown in the following table:
TABLE 1 light and heavy chain CDRs of acridinium ester antibodies
| CDR | Corresponding SEQ ID NO. | Sequence of |
| 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: preparing streptavidin magnetic bead working solution
And finally obtaining 1mg/mL streptavidin magnetic bead working solution according to the instruction operation of the streptavidin magnetic bead kit, and storing the streptavidin magnetic bead working solution in a refrigerator at 4 ℃ for later use.
Example 3: working solution for preparing biotin-labeled PCT (procalcitonin) capture antibody
The biotin-labeled PCT capture antibody was finally obtained at 0.3ug/mL following the protocol of the biotin conjugate kit and stored in a freezer at 4 ℃ until use.
Example 4: working solution for preparing acridinium ester labeled PCT detection antibody
(1) The PCT detection antibody was labeled with acridinium ester according to the procedures of the acridinium ester chemiluminescent labeling kit, wherein the acridinium ester concentration used to label the detection antibody was 0.5mmol/L and 10. mu.L of acridinium ester was used.
(2) After the labeling is finished, the concentration of the PCT detection antibody for labeling the acridinium ester is determined by using an enzyme labeling instrument, then the detection antibody labeled with the acridinium ester is diluted to 0.3ug/mL by using a buffer solution in the acridinium ester chemiluminescence labeling kit, and the diluted detection antibody is stored in a refrigerator at 4 ℃ for later use.
Example 5: test sample
(1) Experiments were carried out with molar ratios of acridinium ester antibody to acridinium ester for labeling of 0.5:1, 1:1, 2:1, 4:1, respectively.
(2) Adding 50 mu L of sample to be detected in a reaction cup, adding 50 mu L of coated biotin-labeled PCT capture antibody, 50 mu L of streptavidin magnetic bead working solution, 50 mu L of acridinium ester antibody and 50 mu L of acridinium ester-labeled PCT detection antibody working solution in the reaction cup in the following sequence, incubating for 10min at 37 ℃, and cleaning.
(3) The reaction cup was placed on a chemiluminescence immunoassay analyzer for testing at a test wavelength of 430 nm.
The results of the detection are shown in tables 2, 3, 4 and 5 (corresponding to molar ratios of acridinium ester antibody to acridinium ester used for labeling of 0.5:1, 1:1, 2:1 and 4:1, respectively).
Table 2 (0.5: 1):
| control group signal value | Signal values of the experimental group | Relative amplitude 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):
| control group signal value | Signal values of the experimental group | Relative amplitude 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):
| control group signal value | Signal values of the experimental group | Relative amplitude 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):
| control group signal value | Signal values of the experimental group | Relative amplitude 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 shows no addition of acridinium ester antibody, and the experimental group shows addition of corresponding molar amount of acridinium ester antibody. S0 indicates no addition of PCT antigen, S1 indicates addition of 0.5ng/mL of PCT antigen, S2 indicates addition of 2ng/mL of PCT antigen, S3 indicates addition of 10ng/mL of PCT antigen, and S4 indicates addition of 50ng/mL of PCT antigen.
Analysis of results
Under the condition of S0, the signal generated at this time was nonspecific adsorption, and the extent of decrease in nonspecific adsorption in the experimental group as compared with the control group was represented by the "relative amplitude" in the table. When the molar ratio of the acridinium ester antibody to the acridinium ester used for labeling is 0.5:1, 1:1, 2:1, or 4:1, non-specific adsorption is reduced by 2.0%, 39.2%, 70.0%, or 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 the acridinium ester antibody to the acridinium ester is 0.5:1, the S1/S0 of the control group is 4.37, and the S1/S0 of the experimental group is 4.29; when the molar ratio of the acridinium ester antibody to the acridinium ester used for labeling is 1:1, the S1/S0 of the control group is 4.37, and the S1/S0 of the experimental group is 6.79; when the molar ratio of the acridinium ester antibody to the acridinium ester used for labeling is 2:1, the S1/S0 of the control group is 4.37, and the S1/S0 of the experimental group is 13.37; when the molar ratio of the acridinium ester antibody to the acridinium ester is 4:1, the S1/S0 of the control group is 4.37, and the S1/S0 of the experimental group is 11.62. It can be seen from the results that when the molar ratio is 2:1, the sensitivity difference between the experimental group and the control group is the largest, indicating that the sensitivity is significantly improved. When the acridinium ester antibody: when the mole 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 shows that the acridinium ester antibody does not influence the linear performance of the reagent in the experiment of detecting the antigen by a chemiluminescence method, and has good practicability, but when the acridinium ester antibody: when the molar ratio of the acridinium ester used for labeling is 4:1, the corresponding relative amplitude of S1-S4 is beyond 10%, which indicates that at the ratio, the acridinium ester antibody can influence the linear function of the reagent in the experiment for detecting the antigen by the chemiluminescence method.
Sequence listing
<110> Nanjing Nodezam medical science and technology Limited
<120> magnetic particle chemiluminescence detection kit and detection method thereof
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Claims (10)
1. A magnetic particle chemiluminescence detection kit comprises an acridinium ester labeled antibody, a capture antibody and magnetic beads, and is characterized by further comprising the acridinium ester antibody.
2. The method 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 acridinium ester antibody and an acridinium ester labeled antibody into the reaction cup.
4. The detection method according to claim 3, wherein the capture antibody is a biotin-labeled PCT capture antibody coated magnetic bead, the magnetic bead is a streptavidin magnetic bead working solution, 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, characterized in that said acridinium ester antibody is selected from monoclonal or polyclonal antibodies, preferably monoclonal antibodies.
7. The kit according to claim 1 or the detection method according to claim 3, wherein the acridinium ester antibody is a monoclonal antibody, whose VL CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID nos. 1-3, respectively, and whose VH CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID nos. 4-6, respectively.
8. 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 for labeling is selected from 0.1-5: 1, preferably 0.5-4: 1, more preferably 1-3: 1, and most preferably 2: 1.
9. An acridinium ester antibody, characterized in that its VL CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NO. 1-3, respectively, and its VH CDR1, CDR2 and CDR3 have the amino acid sequences of SEQ ID NO. 4-6, respectively.
10. Use of the acridinium ester antibody of claim 9 in the preparation of a magnetic particle chemiluminescence detection kit or in a magnetic particle chemiluminescence detection method.
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