WO2023123154A1

WO2023123154A1 - Method for rapidly constructing sandwich elisa

Info

Publication number: WO2023123154A1
Application number: PCT/CN2021/142829
Authority: WO
Inventors: 陈平; 洪伟; 陈爽
Original assignee: 深圳先进技术研究院; 中国科学院深圳理工大学(筹)
Priority date: 2021-12-28
Filing date: 2021-12-30
Publication date: 2023-07-06
Also published as: CN116355086A

Abstract

A method for rapidly constructing a sandwich ELISA, which belongs to the field of biotechnology. In the method, by using a co-immunoprecipitation principle, a known monoclonal antibody is bound to a target antigen to directly screen and obtain a sequence of a target antibody from a polyclonal antibody. The whole process simulates the binding of an antibody and an antigen in a sandwich ELISA. Factors such as overlapping of two antibody recognition domains and interference of a steric hindrance effect between proteins are avoided, time and labor are saved on, and a sandwich ELISA and a corresponding kit are rapidly developed.

Description

A method for rapid construction of sandwich ELISA

technical field

The invention belongs to the field of biotechnology, in particular to a method for rapidly constructing sandwich ELISA.

Background technique

An enzyme-linked immunosorbent assay (ELISA) is a specific type of enzyme immunoassay (EIA) that uses antibodies to quantify a molecule of interest. Antibodies are used to specifically detect analytes (e.g. peptides, proteins, antibodies, small molecules). Enzymes (e.g. horseradish peroxidase, HRP) are directly or indirectly conjugated to this antibody, providing detection and possibly signal amplification. Target molecules can be toxins or other foreign substances that cause the animal's immune system to mount a defensive response, such as influenza viruses or environmental pollutants. The range of potential antigens is wide, so ELISAs are used in many research and assay fields to detect and quantify antigens in a variety of sample types. Using ELISA to analyze specific substances such as cell lysates, blood samples, food, etc., is widely used in scientific research, medical diagnosis, and food hygiene and environmental safety monitoring.

At present, the enzyme-linked adsorption test is divided into: direct ELISA, indirect ELISA, Competitive ELISA and sandwich ELISA. At present, the sensitivity and specificity of sandwich ELISA are superior to other types of ELISA. Sandwich ELISA, commonly known as "sandwich" ELISA, uses the capture antibody coated on the plate to capture the target antigen, and then uses the detection antibody to recognize the antigen, and can quantitatively detect the target molecule in the sample. Currently, in sandwich ELISA, monoclonal antibodies and polyclonal antibodies can be combined with each other. In view of the diversity of components in polyclonal antibodies, resulting in high non-specific signals, sandwich ELISA prefers to use two monoclonal antibodies.

The core technology in the Sandwich ELISA development process is to develop two monoclonal antibodies that can be combined with each other. The current routine development plan is to use hybridoma technology to develop monoclonal antibodies related to multiple target molecules, and then combine them one by one to test the specificity and sensitivity of the combined detection. This process may be due to the overlapping of antibody recognition domains and the impact of steric hindrance effects between proteins, resulting in the failure of the development of ELISA. In addition, from animal immunization, fusion and cell line screening, antibody combination testing, the time and economic cost of the whole process are huge.

technical problem

In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a method for rapidly constructing sandwich ELISA, which can effectively avoid the time-consuming and laborious construction due to factors such as the overlap of the recognition domains of the two antibodies and the interference of steric effects between proteins. The problem.

technical solution

The invention discloses a method for rapidly constructing a sandwich ELISA, which comprises: screening polyclonal antibodies of known antigens through co-immunoprecipitation, which can form an immunoenzyme-linked adsorption assay double antibody with a monoclonal antibody of a known target antigen. Anti-sandwich paired antibody sequences, and then use prokaryotic expression or protein synthesis methods to obtain the target antibody to complete the rapid construction of sandwich ELISA.

Preferably, the method for rapidly constructing sandwich ELISA comprises the following steps:

1) Incubate the biotin-labeled monoclonal antibody with the antigen overnight to form a stable antibody-antigen complex;

2) Add the polyclonal antibody to be screened to the antibody-antigen complex obtained in step 1), and continue to incubate to form a complex similar to a "sandwich" structure;

3) Add the streptomycin-labeled solid medium to the reaction system in step 2), continue to incubate, and separate the complexes similar to the "sandwich" structure by centrifugation to obtain an antibody that can bind to the antigen together with the monoclonal antibody, sequence, and then Through expression and purification, the target antibody is obtained.

Further preferably, in step 1), the molar ratio of monoclonal antibody to antigen is 1:1.

Further preferably, the molar concentration of the biotin-labeled monoclonal antibody used is greater than that of the polyclonal antibody.

Further preferably, in step 3), the solid medium adopts gel particles.

More preferably, the gel particles are blocked with 1%-3% bovine serum albumin before use.

Further preferably, 1%-3% bovine serum albumin is prepared by adding 0.1-0.3 mg of BSA powder to 10 ml of PBS-T solution.

Further preferably, in step 3), the specific operation of sequencing is as follows:

First wash the gel particles, use ammonia water to elute the positive antibody on the gel particles, freeze-dry the eluted liquid, recover the gel strip containing the target band by gel electrophoresis, perform mass spectrometry sequencing, and obtain the light weight of the target antibody chain variable region sequence.

Still further preferably, the gel particles are washed with 150 mM ammonium hydrogen phosphate with a pH value of 7.4, and the positive antibodies on the gel particles are eluted with 150 mM ammonia water.

Further preferably, in step 3), the specific operations of expression and purification are as follows:

The sequence obtained by sequencing was cloned into a plasmid vector by gene synthesis, and a His tag was added to the N segment of the sequence at the same time, and the synthesized plasmid was transformed into E. coli expression bacteria, induced by IPTG, and the induced bacterial liquid The total protein was obtained after crushing, and the target protein was purified by His tag purification method.

Beneficial effect

The method disclosed in the present invention uses the principle of co-immunoprecipitation, uses known monoclonal antibodies to bind the target antigen, directly screens and obtains the target antibody sequence in the polyclonal antibody, and the whole process simulates the binding of the sandwich ELISA antibody antigen, directly in the polyclonal antibody stage The pairing of the two antibodies avoids the overlap of the recognition domains of the two antibodies and the interference of the steric effect between the proteins, thus saving the time and economic cost required for the development of monoclonal antibodies, and can save time and effort for rapid development Sandwich ELISA and corresponding kits. The specific advantages are reflected in:

First, the present invention can save the time and cost of developing sandwich ELISA. When the target antigen already has monoclonal antibody and polyclonal antibody, the method of immunoprecipitation can be directly used for antibody combination, and a suitable antibody combination can be obtained by screening. It takes about 2-3 hours before and after development month;

Second, the present invention can save the antigen cost of developing sandwich ELISA. The biggest bottleneck in the development of sandwich ELISA for rare proteins is the lack of target protein, which makes it difficult to develop antibodies. The present invention only needs to use about 100ug of antigen to develop sandwich ELISA;

Third, the present invention can save the economic cost of developing sandwich ELISA. The development of conventional methods requires a large amount of economic cost for antibody development, but the present invention uses low amounts of antigens and antibodies used in the process; in addition In addition, mass spectrometry sequencing, gene synthesis and prokaryotic expression are all routine experimental operations with low economic costs.

Further, the double-antibody and antigen sandwich complexes are separated from the solution by using labeled gel particles or other solid media.

Further, BSA solution with different concentrations was used to block and PBST solution to wash to reduce non-specific adsorption in co-immunoprecipitation.

Further, ammonia water is used to elute the bound antibody-antigen complex, which facilitates direct freeze-drying and sequencing in the later stage, while avoiding a large amount of damage to the combination of biotin and streptomycin.

Description of drawings

Fig. 1 is a flow chart of the technical solution of the present invention;

Fig. 2 is the flowchart of the first part of co-immunoprecipitation;

Figure 3 is a plasmid vector with His-tag pET-30a (+) added to the N section;

Figure 4 is a comparison result of western blot between the experimental group and the control group after co-immunoprecipitation;

Figure 5 is a comparison result of Coomassie Brilliant Blue staining between the experimental group and the control group after co-immunoprecipitation;

Figure 6 is the analysis result of the sequencing result;

Figure 7 is the result of prokaryotic expression;

Figure 8 is the result of indirect ELISA.

Embodiments of the present invention

In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only It is an embodiment of a part of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

It should be noted that the terms "first" and "second" in the description and claims of the present invention and the above drawings are used to distinguish similar objects, but not necessarily used to describe a specific sequence or sequence. It is to be understood that the data so used are interchangeable under appropriate circumstances such that the embodiments of the invention described herein can be practiced in sequences other than those illustrated or described herein. Furthermore, the terms "comprising" and "having", as well as any variations thereof, are intended to cover a non-exclusive inclusion, for example, a process, method, system, product or device comprising a sequence of steps or elements is not necessarily limited to the expressly listed instead, may include other steps or elements not explicitly listed or inherent to the process, method, product or apparatus.

The present invention is described in further detail below in conjunction with accompanying drawing:

As the flow chart shown in accompanying drawing 1, the technical solution of the invention is divided into four parts:

The first part: co-immunoprecipitation, screening of suitable polyclonal antibodies, the specific steps are shown in Figure 2, specifically including the following steps:

1) First, incubate the biotin-labeled monoclonal antibody (1.33mM) with the antigen Aβ1-42 (1.35mM) in 100ul of phosphate buffer for four overnight to form a stable antibody-antigen complex. In order to improve the efficiency, the molar ratio of monoclonal antibody to antigen is close to 1:1;

2) Then add the polyclonal antibody (1.13mM) to be screened to the antibody-antigen complex incubated in the previous step, and incubate in 100 μL of phosphate buffer at room temperature for two hours, and a complex similar to a "sandwich" structure has been formed. To prevent the polyclonal antibody from competing with the labeled monoclonal antibody from the antigen binding site, ensure that the molar concentration of the biotinylated monoclonal antibody is greater than that of the polyclonal antibody.

3) Add the streptomycin-labeled gel particles to the solution in the previous step, incubate in 1ml of phosphate buffer for 1.5 hours at room temperature, and screen out the bound positive antibodies by centrifugation. During the incubation, be careful Shake the sample gently to avoid sedimentation of the gel particles. In order to avoid false positives caused by non-specific adsorption, the gel particles need to be blocked with bovine serum albumin ranging from 1% to 3%, and the precipitate after centrifugation should be washed with PBS-T (0.05% tween). Three times, at the same time, it is necessary to set up a negative control (no antigen is added during the whole co-immunoprecipitation process), so as to facilitate the elimination of false positive sequences in the later sequencing results.

Part II: Antibody Sequencing and Data Analysis

Include the following steps:

1) First, wash the gel particles with 1 mL of 150 mM ammonium bicarbonate (pH = 7.4) to wash away the residual PBST.

2) Use 150mM ammonia water to elute the positive antibodies on the gel particles. Because 150mM ammonia water is difficult to destroy the connection between biotin and streptomycin, most of the eluted antibodies are positive antibodies obtained by screening.

3) The eluted liquid was lyophilized and run on SDS-PAGE non-reducing gel.

4) Recover the gel strip containing the target band and perform mass spectrometry sequencing to obtain the light chain variable region sequence of the target antibody.

Part III: Expression and Purification of Antibodies

Include the following steps:

1) The sequence obtained by sequencing was cloned into the pET-30a(+) plasmid vector by gene synthesis, and a His tag was added to the N segment of the sequence, as shown in Figure 3;

2) Transform the synthesized plasmid into Escherichia coli expression bacteria (BL21 strain), and use IPTG to induce. The induction conditions (time, temperature, IPTG concentration) depend on the protein;

3) The induced bacterial solution is processed by ultrasonication or high-pressure disruption to obtain the total protein, and the target protein is purified by using the purification strategy of the His tag.

Part IV: Result verification: use ELISA to verify the purified antibody

Include the following steps:

1) Use indirect ELISA to verify the affinity of the purified antibody to the antigen;

2) Use sandwich ELISA to verify the detection sensitivity of the paired antibodies.

Specifically, the present invention has been confirmed to be feasible through preliminary simulation experiments, including the following steps:

1) Co-immunoprecipitation

After the biotin-labeled Aβ1-42 monoclonal antibody 4G8 (1.33mM) and Aβ1-42 (1.35mM) were incubated in 100 μL of phosphate buffer to form a complex, the polyclonal antibody S98 (1.13 mM) to form a “sandwich” complex; use streptomycin-labeled gel particles to separate the “sandwich” complex from the solution. In order to avoid the impact of non-specific adsorption, a control group (without adding Aβ1 -42) as well as blocking with BSA and washing the gel particles with PBST, and then eluting the gel particles with ammonia water, using western Validation by blot and Coomassie brilliant blue staining.

The results are shown in Figure 4 and Figure 5. In Figure 4, the results of immunoprecipitation were verified by western blot. After co-immunoprecipitation, the experimental group and the control group western Blot comparison: the experimental group screened out the S98 antibody that can successfully pair with 4G8, and the control group had no obvious antibody bands. Therefore, it can be seen that the experimental group can specifically screen out suitable antibodies, and it can be seen that the influence of non-specific adsorption is extremely low in the control group.

In Figure 5, the Coomassie Brilliant Blue staining of the experimental group and the control group after co-immunoprecipitation was compared, and the supernatant was stained with Coomassie Brilliant Blue after the separated gel particles were eluted with ammonia water. In Figure 5 (a) the experimental group screened out the S98 antibody that could successfully pair with 4G8, and the control group had no obvious antibody bands; Figure 5 (b) shows that comparing the supernatant and gel particles after elution, most of the biological Primer-labeled 4G8 (4G8b) did not elute.

2) Sequencing and data analysis

The gel strips of the experimental group and the control group were sent for mass spectrometry, and later combined with Uniprot, IMGT database and sequence comparison tools for analysis to find out the complementarity determining region (CDR) and framework region (FR) of the light chain variable region, The data result is shown in Figure 6.

3) Gene synthesis and prokaryotic expression

The target sequence obtained by sequencing was cloned into the plasmid vector of pET-30a(+) (as shown in Figure 3), expressed in the expression bacteria of BL21, and purified by His-tag in the later stage, the result is shown in Figure 7, the result of prokaryotic expression It shows that the sequence of one of them is cloned into the vector, and it can be expressed in large quantities in Escherichia coli.

4) Result verification: use ELISA to verify the purified antibody

The results of the indirect ELISA are shown in Figure 8. It can be seen that in the experimental group, the VL fragment expressed in prokaryotes can recognize the antigen, and has a stronger affinity with the antigen than the negative control.

In summary, the present invention intends to use the monoclonal antibody of the known target antigen to find out the polyclonal antibody of the known antigen through the principle of co-immunoprecipitation, which can form a double antibody for immunoenzyme-linked adsorption experiment with the known monoclonal antibody. Sandwich paired antibody sequences, using prokaryotic expression or protein synthesis experimental methods to obtain the target antibody, and then achieve the goal of developing sandwich ELISA.

The present invention can still be used to develop monoclonal antibodies. Through the principle of co-immunoprecipitation, monoclonal antibodies of known target antigens are used to find polyclonal antibodies of known antigens that can form immunoenzyme-linked adsorption with known monoclonal antibodies. Experiment with the antibody sequence of the double-antibody sandwich pairing, and use antibody engineering to obtain conventional monoclonal antibodies or humanized antibodies.

The above content is only to illustrate the technical ideas of the present invention, and cannot limit the protection scope of the present invention. Any changes made on the basis of the technical solutions according to the technical ideas proposed in the present invention shall fall within the scope of the claims of the present invention. within the scope of protection.

Claims

A quick way to build sandwiches The ELISA method is characterized in that it comprises: by co-immunoprecipitation, among the polyclonal antibodies of known antigens, screening out the ones that can form the double-antibody sandwich pairing of the immunoenzyme-linked adsorption experiment with the monoclonal antibodies of the known target antigens Antibody sequence, and then use the method of prokaryotic expression or protein synthesis to obtain the target antibody to complete the rapid construction of sandwich ELISA.
The method for rapidly constructing sandwich ELISA according to claim 1, is characterized in that, comprises the following steps:

1) Incubate the biotin-labeled monoclonal antibody with the antigen overnight to form a stable antibody-antigen complex;

2) Add the polyclonal antibody to be screened to the antibody-antigen complex obtained in step 1), and continue to incubate to form a complex similar to a "sandwich" structure;

3) Add the streptomycin-labeled solid medium to the reaction system in step 2), continue to incubate, and separate the complex similar to the "sandwich" structure by centrifugation to obtain an antibody that can bind to the antigen together with the monoclonal antibody, sequence, and then Through expression and purification, the target antibody is obtained.
The method for rapidly constructing sandwich ELISA according to claim 2, characterized in that, in step 1), the molar ratio of monoclonal antibody to antigen is 1:1.
The method for rapidly constructing a sandwich ELISA according to claim 2, wherein the molar concentration of the biotinylated monoclonal antibody used is greater than that of the polyclonal antibody.
The method for rapidly constructing sandwich ELISA according to claim 2, characterized in that in step 3), the solid medium is gel particles.
The method for quickly constructing a sandwich ELISA according to claim 5, wherein the gel particles are blocked with 1% to 3% bovine serum albumin before use.
The method for quickly constructing a sandwich ELISA according to claim 5, characterized in that, in step 3), the specific operations of sequencing are as follows:

First wash the gel particles, use ammonia water to elute the positive antibody on the gel particles, freeze-dry the eluted liquid, recover the gel strip containing the target band by gel electrophoresis, perform mass spectrometry sequencing, and obtain the target antibody light chain variable region sequence.
The method for rapidly constructing a sandwich ELISA according to claim 7, wherein the gel particles are washed with 150 mM ammonium hydrogen phosphate with a pH value of 7.4, and the positive antibodies on the gel particles are eluted with 150 mM ammonia water.
The method for quickly constructing a sandwich ELISA according to claim 2, characterized in that, in step 3), the specific operations of expression and purification are as follows:

The sequence obtained by sequencing was cloned into a plasmid vector by gene synthesis, and a His tag was added to the N segment of the sequence at the same time, and the synthesized plasmid was transformed into E. coli expression bacteria, induced by IPTG, and the induced bacterial liquid The total protein was obtained after crushing, and the target protein was purified by His tag purification method.