Chemiluminescent screening method for specific hybridoma cells based on proximity-induced immunoassay

Abstract

The present invention relates to a specific hybridoma cell chemiluminescence screening method based on ortho-induced immunoassay. The detection solution of the chemiluminescence screening method includes a pair of antigen-DNA conjugates, a double-stranded DNA probe, a template DNA, a deoxyribonucleoside triphosphate, a nucleic acid ligase and a polymerase. After the hybridoma single cells distributed in a 96-well plate are cultured for 1 day, the supernatant is taken to react with the detection solution. If it is a specific hybridoma cell, the supernatant will contain its secreted specific antibodies, which can be combined with the antigen-DNA conjugate pair in the detection solution through immune recognition, and then based on the ortho effect, the nucleic acid assembly and rolling circle amplification reaction are triggered to produce a large amount of pyrophosphate ions (PPi). PPi can complex horseradish peroxidase-Cu 2+ Complex (HRP‑Cu 2+ ) 2+ Restore the activity of HRP. Use the chemiluminescent signal generated by HRP catalysis to quantitatively analyze the secreted specific antibodies and complete the screening of specific hybridoma cells.

Description

1. Technical Field

[0001] The present invention introduces a method for chemiluminescent screening of specific hybridoma cells based on proximity-induced immunoassay. The method performs proximity-induced immunoassay on specific antibodies secreted by hybridoma cells, combines DNA assembly and rolling circle amplification reaction, and uses pyrophosphate ions (PPi) to 2+ The strong complexation of HRP can restore the HRP enzyme activity to produce chemiluminescent signals, thus achieving simple, sensitive and rapid screening of specific hybridoma cells. 2. Background Technology

[0002] Hybridoma technology is a well-known method for producing monoclonal antibodies. Since hybridoma technology includes animal immunization, cell fusion, screening and subcloning of specific hybridoma cells, antibody characterization and other parts, the production process of monoclonal antibodies is time-consuming and cumbersome, resulting in low monoclonal antibody drug production, high prices, and difficulty in popularization. The lengthy time of hybridoma technology is mainly reflected in the screening of specific hybridoma cells. The most common screening method for hybridoma cells today is enzyme-linked immunosorbent assay (ELISA). Since the detection sensitivity of ELISA is not high, it takes a long time for cell cloning (1-2 weeks) to obtain high concentrations of monoclonal antibodies for detection. In order to achieve rapid screening, recently, based on microdroplet technology, microdroplets encapsulating single hybridoma cells have been prepared. Due to the small volume of microdroplets (reaching nanoliters), after several hours of culture, the concentration of specific antibodies secreted in the microdroplets can reach μg / mL, which can be detected by sandwich fluorescent immunoassay and achieve rapid screening of specific hybridoma cells. However, the instruments required for microdroplet technology are expensive, the operation is precise, professional experimental personnel are required, and the single cell coverage rate is low, which is not suitable for large-scale and low-cost production of monoclonal antibodies.

[0003] Homogeneous immunoassay has always been the development direction of convenient immunoassay methods because it does not require separation, multi-step reactions, and multi-step washing operations. Proximity-induced immunoassay is a homogeneous immunoassay method developed in recent years. It uses a pair of antibody-DNA (or antigen-DNA) conjugate probes to immune-recognize the target antigen (or antibody), and further induces DNA assembly to generate signals through the proximity effect. The proximity-induced immunoassay method converts the detection of antigens (or antibodies) into the detection of DNA. By combining with various DNA signal amplification technologies, ultra-sensitive detection of antigens (or antibodies) can be achieved.

[0004] The present invention designs antigen-DNA conjugate probes for specific antibodies, combines the adjacent induction effect, nucleic acid rolling circle amplification reaction and PPi to Cu 2+A specific hybridoma cell chemiluminescence screening method based on ortho-induced immunoassay was developed based on the strong complexation reaction of the chemiluminescent reaction. The method can achieve specific screening of hybridoma cells distributed in a 96-well plate by limiting dilution method within 1 day. The screening method proposed by the present invention is simple, rapid and universal, and has great application prospects in hybridoma technology and clinical applications. III. Summary of the invention

[0005] The present invention is to prepare antigen-DNA conjugate probes for specific antibodies based on the adjacent induction effect, rolling circle amplification reaction and PPi to Cu 2+ A signal amplification strategy was designed based on the complex reaction to develop a highly sensitive homogeneous antibody detection method, which was applied to the rapid screening of specific hybridoma cells.

[0006] The present invention is achieved through the following technical solutions:

[0007] The antigen-DNA1 conjugate and antigen-DNA2 conjugate capable of immune recognition of specific antibodies are synthesized, and the primer chain DNA is hybridized with the closed chain DNA to form a double-stranded DNA probe. The above-prepared pair of antigen-DNA conjugates and double-stranded DNA probes are mixed with template DNA, deoxyribonucleoside triphosphates (dNTPs), nucleic acid ligase SplintR, and polymerase phi29 to serve as a detection solution; hybridoma cells are distributed in a 96-well plate by limiting dilution method, and after culturing for 1 day, the supernatant is taken as a sample solution; the above detection solution and sample If there are specific antibodies secreted by hybridoma cells in the sample solution, they will be recognized and bound by antigen-DNA1 conjugate and antigen-DNA2 conjugate at the same time, so that DNA1 and DNA2 are close to each other to form a complex, which can hybridize with the closed chain DNA in the double-stranded DNA probe to release the primer chain DNA. The primer chain DNA can bind to the template DNA and perform rolling circle amplification under the action of SplintR and phi29 enzymes, and a large amount of PPi will be generated at the same time. Since PPi can complex horseradish peroxidase-Cu 2+ Complex (HRP-Cu 2+ ) 2+ Restore HRP activity (eg Figure 1 ), so the chemiluminescent signal produced by the HRP-catalyzed reaction of luminol and hydrogen peroxide is used to achieve quantitative detection of secreted specific antibodies and screening of specific hybridoma cells.

[0008] In the above double-stranded DNA probe, the blocking strand DNA is longer than the primer strand DNA, wherein the primer strand DNA is completely hybridized.

[0009] The principle of this method for screening specific hybridoma cells:

[0010] The present invention uses the specific antibodies secreted by hybridoma cells as marker molecules, and realizes the screening of specific hybridoma cells by performing high-sensitivity chemiluminescence imaging detection on the specific antibodies in the single cell culture fluid. Figure 2 ); hybridoma cells are distributed in a 96-well plate by a limiting dilution method as single cells. After one day of culture, although almost no cell clones are still in a single-cell state, trace amounts of specific antibodies secreted by the specific hybridoma cells will be present in the culture fluid; the specific antibodies are detected by an ortho-induced chemiluminescent immunoassay strategy. Since this strategy uses a large amount of PPi generated in rolling circle amplification to restore HRP activity and catalyze the generation of chemiluminescent signals, the detection sensitivity of the antibodies is very high; therefore, the method of the present invention achieves the screening of specific hybridoma cells on a 96-well plate within one day through ultra-sensitive chemiluminescent detection of specific antibodies.

[0011] Compared with the prior art, the present invention has the following characteristics:

[0012] The present invention combines proximity-induced assembly, rolling circle amplification and complexation reaction to design a signal cascade amplification strategy, and develops a specific hybridoma cell chemiluminescence screening method based on proximity-induced immunoassay. Compared with the conventional ELISA screening method, it has the following characteristics:

[0013] (1) This method is based on adjacent site induced immune analysis, can be operated on a 96-well plate and does not require steps such as washing, separation, and drying. It is simple to operate and low in cost.

[0014] (2) Based on the complexation reaction and enzyme activity recovery, a signal switch was designed and combined with rolling circle amplification to develop a new signal amplification strategy to improve the detection sensitivity of specific antibodies. The detection limit reached the fM level, which can realize the detection of antibodies secreted by single cells.

[0015] (3) This method does not require cell cloning and can complete the screening of specific hybridoma cells within one day. IV. Description of the drawings

[0016] Figure 1 .Schematic diagram of detection of specific antibodies by proximity-induced immunoassay

[0017] Figure 2 .Schematic diagram of chemiluminescent screening of specific hybridoma cells V. Specific implementation methods

[0018] Embodiment 1: Combination Figure 1 and 2 , illustrating the invented chemiluminescent screening method for screening 6A6 hybridoma cells that can specifically secrete PCSK9 monoclonal antibodies.

[0019] (1) Hybridoma cells were distributed into a 96-well plate by limiting dilution method, 200 μL of DMEM cell culture medium was added to each well, and the plates were cultured at 37°C in an environment of 5% CO2 and 95% humidity for 24 hours. 10 μL of the supernatant was taken as the sample solution.

[0020] (2) The above 10 μL sample solution was mixed with 10 μL detection solution (containing a certain concentration of antigen-DNA conjugate, double-stranded DNA probe, template DNA, dNTPs, nucleic acid ligase SplintR and polymerase phi29) and reacted at 37°C for 1 hour, followed by the addition of 10 μL 1 μg / mL HRP-Cu 2+ Solution, react at room temperature for 5 minutes;

[0021] (3) Add 20 μL of chemiluminescent substrate solution (containing 2.5 mM H2O2 and 0.5 mM luminol) to the above solution, and immediately use a chemiluminescence imager to collect the chemiluminescence intensity value (exposure for 1 minute). Based on the standard curve of PCSK9 antibodies according to the method of the present invention, the PCSK9 antibodies in the sample solution are determined, and the screening of 6A6 hybridoma cells is achieved.

Claims

1. A method for screening specific hybridoma cells by chemiluminescence based on proximity-induced immunoassay, characterized in that The screening method uses a solution containing antigen-DNA1 conjugate, antigen-DNA2 conjugate, double-stranded DNA probe, template DNA, deoxyribonucleoside triphosphates (dNTPs), nucleic acid ligase SplintR and polymerase phi29 as a detection solution, and uses the supernatant of hybridoma cells after culturing for one day as a sample solution. When the hybridoma cells are specific hybridoma cells, specific antibodies will exist in the sample solution, which can be immunologically bound by the antigen-DNA1 conjugate and the antigen-DNA2 conjugate at the same time. Based on the adjacent effect, DNA1 and DNA2 are hybridized and assembled and hybridized with the closed chain DNA in the double-stranded DNA probe, thereby releasing the primer chain DNA and initiating a rolling circle amplification reaction. The large amount of pyrophosphate ions (PPi) generated can complex the horseradish peroxidase-Cu 2+ Complex (HRP-Cu 2+ ) 2+ , thereby restoring the activity of HRP, catalyzing the reaction of luminol and hydrogen peroxide to produce chemiluminescent signals, achieving quantitative analysis of specific antibodies and screening of specific hybridoma cells, and including the following detection steps: Step 1: The hybridoma cells were distributed in a 96-well plate by limiting dilution method. After 1 day of incubation, the supernatant was mixed with the detection solution and reacted at 37°C for 1 hour. Step 2: Add HRP-Cu 2+ After 5 minutes of reaction, a mixture of luminol and hydrogen peroxide was added, and the chemiluminescence intensity was immediately measured using a chemiluminescence imaging instrument. Step 3: Use the standard curve method to determine the concentration of specific antibodies in the supernatant, and then screen out specific hybridoma cells.

2. The screening method according to claim 1, characterized in that The double-stranded DNA probe is a hybrid of a long closed-strand DNA and a short primer-strand DNA.

3. The screening method according to claim 1, characterized in that The DNA1 and DNA2 can be hybridized and assembled under the proximity effect and further hybridized with the closed chain DNA to release the primer chain DNA.

4. The screening method according to claim 1, characterized in that The primer strand DNA can hybridize with the template DNA and form a circular template DNA under the action of nucleic acid ligase SplintR.

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